Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof

ABSTRACT

The present disclosure provides methods and compositions for genetically modifying lymphocytes and related methods that include genetically modifying T cells and/or NK cells. The methods use replication incompetent recombinant retroviral particles that comprise a pseudotyping element on their surface and optionally a membrane-bound T cell activation element, such as an anti-CD3, and encode one or more engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR). The methods can include contacting PBMCs with replication incompetent recombinant retroviral particles for various exemplary time periods, such as less than 24 hours or in some illustrative embodiments less than 15 minutes. In some aspects, the present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in whole blood or a component thereof. In some embodiments a lymphodepletion filter assembly is used before or after forming a reaction mixture where lymphocytes are contacted with recombinant retroviral particles in a closed system, to genetically modify the lymphocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.15/462,855 filed Mar. 19, 2017; U.S. application Ser. No. 15/644,778filed Jul. 8, 2017; U.S. application Ser. No. 16/490,201 filed Mar. 3,2018; International Application No. PCT/US2018/051392 filed Sep. 17,2018; and International Application No. PCT/US2019/49259, filed Sep. 2,2019; and claims the benefit of U.S. Provisional Application No.62/821,434, filed Mar. 20, 2019; and U.S. Provisional Application No.62/894,853, filed Sep. 1, 2019; and U.S. application Ser. No. 15/462,855claims the benefit of U.S. Provisional Application No. 62/390,093, filedMar. 19, 2016; U.S. Provisional Application No. 62/360,041, filed Jul.8, 2016; and U.S. Provisional Application No. 62/467,039, filed Mar. 3,2017; and U.S. application Ser. No. 15/644,778 is a continuation-in-partof International Application No. PCT/US2017/023112, filed Mar. 19, 2017;and a continuation-in-part of U.S. patent application Ser. No.15/462,855, filed Mar. 19, 2017; and claims the benefit of U.S.Provisional Application No. 62/360,041, filed Jul. 8, 2016, and U.S.Provisional Application No. 62/467,039, filed Mar. 3, 2017; U.S.application Ser. No. 16/490,201 is a National Stage of InternationalApplication No. PCT/US2018/020818, filed Mar. 3, 2018; and claims thebenefit of U.S. Provisional Application No. 62/560,176, filed Sep. 18,2017; U.S. Provisional Application No. 62/564,253, filed Sep. 27, 2017;U.S. Provisional Application No. 62/564,991, filed Sep. 28, 2017; andU.S. Provisional Application No. 62/728,056, filed Sep. 6, 2018; andInternational Application No. PCT/US2018/051392 is acontinuation-in-part of International Application No. PCT/US2018/020818,filed Mar. 3, 2018; and claims the benefit of U.S. ProvisionalApplication No. 62/560,176, filed Sep. 18, 2017; U.S. ProvisionalApplication No. 62/564,253, filed Sep. 27, 2017; U.S. ProvisionalApplication No. 62/564,991, filed Sep. 28, 2017; and U.S. ProvisionalApplication No. 62/728,056, filed Sep. 6, 2018; and InternationalApplication No. PCT/US2019/49259 is a continuation-in-part ofInternational Application No. PCT/US2018/051392 filed Sep. 17, 2018; andclaims the benefit of U.S. Provisional Application No. 62/726,293, filedSep. 2, 2018; U.S. Provisional Application No. 62/726,294, filed Sep. 2,2018; U.S. Provisional Application No. 62/728,056 filed Sep. 6, 2018;U.S. Provisional Application No. 62/732,528, filed Sep. 17, 2018, U.S.Provisional Application No. 62/821,434, filed Mar. 20, 2019; and U.S.Provisional Application No. 62/894,853, filed Sep. 1, 2019;International Application No. PCT/US2018/020818 is acontinuation-in-part of International Application No. PCT/US2017/023112filed Mar. 19, 2017; a continuation-in-part of International ApplicationNo. PCT/US2017/041277 filed Jul. 8, 2017; a continuation-in-part of U.S.application Ser. No. 15/462,855 filed Mar. 19, 2017; and acontinuation-in-part of U.S. application Ser. No. 15/644,778 filed Jul.8, 2017; and claims the benefit of U.S. Provisional Application No.62/467,039 filed Mar. 3, 2017; U.S. Provisional Application No.62/560,176 filed Sep. 18, 2017; U.S. Provisional Application No.62/564,253 filed Sep. 27, 2017; and U.S. Provisional Application No.62/564,991 filed Sep. 28, 2017; International Application No.PCT/US2017/023112 claims the benefit of U.S. Provisional Application No.62/390,093, filed Mar. 19, 2016; U.S. Provisional Application No.62/360,041, filed Jul. 8, 2016; and U.S. Provisional Application No.62/467,039, filed Mar. 3, 2017; International Application No.PCT/US2017/041277 claims the benefit of International Application No.PCT/US2017/023112, filed Mar. 19, 2017; U.S. patent application Ser. No.15/462,855, filed Mar. 19, 2017; U.S. Provisional Application No.62/360,041, filed Jul. 8, 2016; and U.S. Provisional Application No.62/467,039, filed Mar. 3, 2017. These applications are incorporated byreference herein in their entireties.

SEQUENCE LISTING

This application hereby incorporates by reference the material of theelectronic Sequencing Listing filed concurrently herewith. The materialsin the electronic Sequence Listing is submitted as a text (.txt) fileentitled “F1_001_US_04_Sequence_Listing_March_18_2020.txt” created onMar. 18, 2020 which has a file size of 460,165 bytes, and is hereinincorporated by reference in its entirety.

FIELD OF INVENTION

This disclosure relates to the field of immunology, or morespecifically, to the genetic modification of T lymphocytes or otherimmune cells, and methods of controlling proliferation of such cells.

BACKGROUND OF THE DISCLOSURE

Lymphocytes isolated from a subject (e.g. patient) can be activated invitro and genetically modified to express synthetic proteins that enableredirected engagement with other cells and environments based upon thegenetic programs incorporated. Examples of such synthetic proteinsinclude recombinant T cell receptors (TCRs) and chimeric antigenreceptors (CARs). One CAR that is currently used is a fusion of anextracellular recognition domain (e.g., an antigen-binding domain), atransmembrane domain, and one or more intracellular signaling domainsencoded by a replication incompetent recombinant retrovirus.

While recombinant retroviruses have shown efficacy in infectingnon-dividing cells, resting CD4 and CD8 lymphocytes are refractory togenetic transduction by these vectors. To overcome this difficulty,these cells are typically activated in vitro using stimulation reagentsbefore genetic modification with the CAR gene vector can occur.Following stimulation and transduction, the genetically modified cellsare expanded in vitro and subsequently reintroduced into alymphodepleted patient. Upon antigen engagement in vivo, theintracellular signaling portion of the CAR can initiate anactivation-related response in an immune cell and release of cytolyticmolecules to induce target cell death.

Such current methods requite extensive manipulation and manufacturing ofproliferating T cells outside the body prior to their reinfusion intothe patient, as well as lymphodepleting chemotherapy to free cytokinesand deplete competing receptors to facilitate T cell engraftment. SuchCAR therapies further cannot be controlled for propagation rate in vivoonce introduced into the body, nor safely directed towards targets thatare also expressed outside the tumor. As a result, CAR therapies todayare typically infused from cells expanded ex vivo from 12 to 28 daysusing doses from 1×10⁵ to 1×10⁸ cells/kg and are directed towardstargets, for example tumor targets, for which off tumor on targettoxicity is generally acceptable. These relatively long ex vivoexpansion times create issues of cell viability and sterility, as wellas sample identity in addition to challenges of scalability. Thus, thereare significant needs for a safer, more effective scalable T cell or NKcell therapy.

Since our understanding of processes that drive transduction,proliferation and survival of lymphocytes is central to variouspotential commercial uses that involve immunological processes, there isa need for improved methods and compositions for studying lymphocytes.For example, it would be helpful to identify methods and compositionsthat can be used to better characterize and understand how lymphocytescan be genetically modified and the factors that influence theirsurvival and proliferation. Furthermore, it would be helpful to identifycompositions that drive lymphocyte proliferation and survival. Suchcompositions could be used to study the regulation of such processes. Inaddition to methods and compositions for studying lymphocytes, there isa need for improved vital packaging cell lines and methods of making andusing the same. For example, such cell lines and methods would be usefulin analyzing different components of recombinant viruses, such asrecombinant retroviral particles, and for methods that use packagingcells lines for the production of recombinant retroviral particles.

More recent methods have been developed that can be performed withoutpre-activation and ex vivo expansion. However, further reduction in thecomplexity and time required for such methods would be highly desirable,especially if such methods allow a subject to have their bloodcollected, for example within an infusion center, and then reintroducedinto the subject that same day. Furthermore, simpler and quicker methodsalone or methods that require fewer specialized instruments, coulddemocratize these cell therapy processes, which are currently performedregularly only at highly specialized medical centers.

Some groups have attempted to simplify ex-vivo processing for celltherapy by eliminating ex-vivo transduction expansion, by infusion viralparticles intravenously, to transduce cells in vivo. However, suchmethods require large quantities of vector and the methods have the riskof inactivation of the retroviral particles by clotting factors, and/orother enzymes present in vivo. Finally, such methods risk a high levelof transduction of non-target cells/organs.

SUMMARY

Provided herein are methods, compositions, and kits that help overcomeissues related to the effectiveness and safety of methods fortransducing and/or genetically modifying lymphocytes such as T cellsand/or NK cells. Certain embodiments of such methods are useful forperforming adoptive cell therapy with these cells. Accordingly, in someaspects, provided herein are methods, compositions, and kits forgenetically modifying lymphocytes, especially T cell and/or NK cells,and/or for regulating the activity of transduced and/or geneticallymodified T cells and/or NK cells. Such methods, compositions, and kitsprovide improved efficacy and safety over current technologies,especially with respect to T cells and/or NK cells that expressrecombinant T cell receptors (TCRs), chimeric antigen receptors (CARs),and in illustrative embodiments microenvironment restricted biologic(“MRB”) CARs. Transduced and/or genetically modified T cells and/or NKcells that are produced by and/or used in methods provided herein,include functionality and combinations of functionality, in illustrativeembodiments delivered from retroviral (e.g. lentiviral) genomes viaretroviral (e.g. lentiviral) particles, that provide improved featuresfor such cells and for methods that utilize such cells, such as researchmethods, commercial production methods, and adoptive cellular therapy.For example, such cells can be produced in less time ex vivo, and thathave improved growth properties that can be better regulated.

In some aspects, methods are provided for transducing and/or geneticallymodifying lymphocytes such as T cells and/or NK cells, and inillustrative embodiments, ex vivo methods for transducing and/orgenetically modifying resting T cells and/or NK cells. Some of theseaspects can be performed much more quickly titan previous methods, whichcan facilitate more efficient research, more effective commercialproduction, and improved methods of patient care. Methods, compositions,and kits provided herein, can be used as research tools, in commercialproduction, and in adoptive cellular therapy with transduced and/orgenetically modified T cells and/or NK cells expressing a TCR or a CAR.

With respect to methods, uses and compositions provided herein thatrelate to transduction of lymphocytes such as T cells and/or NK cells,methods, and associated uses and compositions, are provide herein thatinclude transduction reactions of enriched PBMCs or transductionreactions without prior PBMC enrichment, such as in whole blood that aresimplified and quicker methods for performing ex-vivo cell processing,for example for CAR-T therapy. Such methods require less specializedinstrumentation and training. Furthermore, such methods reduce the riskof non-targeted cell transduction compared to in vivo transductionmethods. Furthermore, provided herein are methods, uses, andcompositions, including embodiments of the methods immediately above,that include certain target inhibitory RNAs, polypeptidelymphoproliferative elements, and pseudotyping elements that can beoptionally be combined with any other aspects provided herein to providepowerful methods, uses, and compositions for driving expansion oflymphocytes, especially T cells and/or NK cells in vitro, ex vivo, andin vivo.

Further details regarding aspects and embodiments of the presentdisclosure are provided throughout this patent application. Sections andsection headers are for ease of reading and are not intended to limitcombinations of disclosure, such as methods, compositions, and kits orfunctional elements therein across sections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are flowcharts of non-limiting exemplary cell processingworkflows. FIG. 1A is a flow chart of a process that uses a system withPBMC enrichment before the contacting of T cells and NK cells in thePBMCs with retroviral particles. FIG. 1B is a flow chart of a process inwhich no blood cell fractionation or enrichment is performed before Tcells and NK cells in the whole blood are contacted with retroviralparticles, and a PBMC enrichment is performed after transduction.

FIG. 2 is a diagram of a non-limiting exemplary leukodepletion filterassembly (200) with associated blood processing bags, tubes, valves, andfilter enclosure (210) comprising a leukodepletion filter set.

FIGS. 3A and 3B show histograms of experimental results with differentpseudotyping elements. FIG. 3A shows a histogram of tire total number oflive cells per well on Day 6 following transduction. FIG. 3B shows ahistogram of the percent of CD3+ cells transduced as measured by eTAGexpression.

FIGS. 4A and 4B show histograms of experimental results withtransduction reaction mixtures that include whole blood, lentiviralparticles, and anti-coagulants EDTA or heparin, without PBMC enrichmentbefore the reaction mixture was formed. The process was performed bycontacting whole blood for 4 hours with the indicated lentiviralparticle F1-3-23G or F1-3-23GU followed by a density gradientcentrifugation-based PBMC enrichment procedure. FIG. 4A shows ahistogram of the absolute cell number per uL of the live lymphocytepopulation. FIG. 4B shows a histogram of the percentage (%) CD3+eTag+cells in the live lymphocyte population at Day 6 post-transduction.

FIG. 5 is a histogram showing the CD3+FLAG+ cell number per μl ofculture at Day 6 after transduction of unstimulated PBMCs by thedifferent recombinant lentiviral particles at an MOI of 1 for theindicated period of time. F1-3-253 encoded an anti-CD19 CAR and F1-3-451encoded a CLE in addition to the same CAR. The lentiviral particles werepseudotyped with VSV-G [VSV-G] and optionally-displayed UCHT1ScFvFc-GPI[VSV-G+U] as indicated. Samples were treated with dapivirine, aninhibitor of reverse transcription (RT inb) or dolutegravir, aninhibitor to integration (INT Inb), as indicated.

FIG. 6 is a schematic of a non-limiting, exemplary transgene expressioncassette containing a polynucleotide sequence encoding a CAR and acandidate CLE of Libraries analyzed in Example 6.

DEFINITIONS

As used herein, the term “chimeric antigen receptor” or “CAR” or “CARs”refers to engineered receptors, which graft an antigen specificity ontocells, for example T cells, NK cells, macrophages, and stem cells. TheCARs of the invention include at least one antigen-specific targetingregion (ASTR), a transmembrane domain (TM), and an intracellularactivating domain (IAD) and can include a stalk, and one or moreco-stimulatory domains (CSDs). In another embodiment, the CAR is abispecific CAR, which is specific to two different antigens or epitopes.After the ASTR binds specifically to a target antigen, the IAD activatesintracellular signaling. For example, the IAD can redirect T cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofantibodies. The non-MHC-restricted antigen recognition gives T cellsexpressing the CAR the ability to recognize an antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

As used herein, the term “microenvironment” means any portion or regionof a tissue or body that has constant or temporal, physical, or chemicaldifferences from other regions of the tissue or regions of the body. Forexample, a “tumor microenvironment” as used herein refers to theenvironment in which a tumor exists, which is the non-cellular areawithin the tumor and the area directly outside the tumorous tissue butdoes not pertain to the intracellular compartment of the cancer cellitself. The tumor microenvironment can refer to any and all conditionsof the tumor milieu including conditions that create a structural and orfunctional environment for the malignant process to survive and/orexpand and/or spread. For example, the tumor microenvironment caninclude alterations in conditions such as, but not limited to, pressure,temperature, pH, ionic strength, osmotic pressure, osmolality, oxidativestress, concentration of one or more solutes, concentration ofelectrolytes, concentration of glucose, concentration of hyaluronan,concentration of lactic acid or lactate, concentration of albumin,levels of adenosine, levels of R-2-hydroxyglutarate, concentration ofpyruvate, concentration of oxygen, and/or presence of oxidants,reductants, or co-factors, as well as other conditions a skilled artisanwill understand.

As used interchangeably herein, the terms “polynucleotide” and “nucleicacid” refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically-modified,non-natural, or derivatized nucleotide bases.

As used herein, the term “antibody” includes polyclonal and monoclonalantibodies, including intact antibodies and fragments of antibodieswhich retain specific binding to antigen. The antibody fragments can be,but are not limited to, fragment antigen binding (Fab) fragments, Fab′fragments, F(ab′)₂ fragments, Fv fragments, Fab′-SH fragments, (Fab′)₂Fv fragments, Fd fragments, recombinant IgG (rIgG) fragments,single-chain antibody fragments, including single-chain variablefragments (scFv), divalent scFv's, trivalent scFv's, and single domainantibody fragments (e.g., sdAb, sdFv, nanobody). The term includesgenetically engineered and/or otherwise modified forms ofimmunoglobulins, such as intrabodies, peptibodies, chimeric antibodies,single-chain antibodies, fully human antibodies, humanized antibodies,fusion proteins including an antigen-specific targeting region of anantibody and a non-antibody protein, heteroconjugate antibodies,multispecific, e.g., bispecific, antibodies, diabodies, triabodies, andtetrabodies, tandem di-scFv's, and tandem tri-scFv's. Unless otherwisestated, the term “antibody” should be understood to include functionalantibody fragments thereof. The term also includes intact or full-lengthantibodies, including antibodies of any class or sub-class, includingIgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

As used herein, the term “antibody fragment” includes a portion of anintact antibody, for example, the antigen binding or variable region ofan intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fe” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

As used interchangeably herein, tire terms “single-chain Fv,” “scFv,” or“sFv” antibody fragments include the V_(H) and V_(L) domains ofantibody, wherein these domains are present in a single polypeptidechain. In some embodiments, the Fv polypeptide further includes apolypeptide linker or spacer between the V_(H) and V_(L) domains, whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

As used herein, “naturally occurring” VH and VL domains refer to VH andVL domains that have been isolated from a host without further molecularevolution to change their affinities when generated in an scFv formatunder specific conditions such as those disclosed in U.S. Pat. No.8,709,755 B2 and application WO/2016/033331A1.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as adissociation constant (Kd). Affinity can be at least 1-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, at least10-fold greater, at least 20-fold greater, at least 30-fold greater, atleast 40-fold greater, at least 50-fold greater, at least 60-foldgreater, at least 70-fold greater, at least 80-fold greater, at least90-fold greater, at least 100-fold greater, or at least 1000-foldgreater, or more, than the affinity of an antibody for unrelated aminoacid sequences. Affinity of an antibody to a target protein can be, forexample, from about 100 nanomolar (nM) to about 0.1 nM, from about 100nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar(fM) or more. As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. The terms “immunoreactive” and “preferentially binds” are usedinterchangeably herein with respect to antibodies and/or antigen-bindingfragments.

As used herein, the term “binding” refers to a direct associationbetween two molecules, due to, for example, covalent, electrostatic,hydrophobic, and ionic and/or hydrogen-bond interactions, includinginteractions such as salt bridges and water bridges. Non-specificbinding would refer to binding with an affinity of less than about 10⁻⁷M, e.g., binding with an affinity of 10⁻⁶ M, 10⁻⁵M, 10⁻⁴ M, etc.

As used herein, reference to a “cell surface expression system” or “cellsurface display system” refers to the display or expression of a proteinor portion thereof on the surface of a cell. Typically, a cell isgenerated that expresses proteins of interest fused to a cell-surfaceprotein. For example, a protein is expressed as a fusion protein with atransmembrane domain.

As used herein, the term “element” includes polypeptides, includingfusions of polypeptides, regions of polypeptides, and functional mutantsor fragments thereof and polynucleotides, including microRNAs andshRNAs, and functional mutants or fragments thereof.

As used herein, the term “region” is any segment of a polypeptide orpolynucleotide.

As used herein, a “domain” is a region of a polypeptide orpolynucleotide with a functional and/or structural property.

As used herein, the terms “stalk” or “stalk domain” refer to a flexiblepolypeptide connector region providing structural flexibility andspacing to flanking polypeptide regions and can consist of natural orsynthetic polypeptides. A stalk can be derived from a hinge or hingeregion of an immunoglobulin (e.g., IgGl) that is generally defined asstretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec.Immunol., 22:161-206). Hinge regions of other IgG isotypes may bealigned with tire IgG1 sequence by placing the first and last cysteineresidues forming inter-heavy chain disulfide (S—S) bonds in tire samepositions. The stalk may be of natural occurrence or non-naturaloccurrence, including but not limited to an altered hinge region, asdisclosed in U.S. Pat. No. 5,677,425. The stalk can include a completehinge region derived from an antibody of any class or subclass. Thestalk can also include regions derived from CD8, CD28, or otherreceptors that provide a similar function in providing flexibility andspacing to flanking regions.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

As used herein, a “polypeptide” is a single chain of amino acid residueslinked by peptide bonds. A polypeptide does not fold into a fixedstructure nor does it have any posttranslational modification. A“protein” is a polypeptide that folds into a fixed structure.“Polypeptides” and “proteins” are used interchangeably herein.

As used herein, a polypeptide may be “purified” to remove contaminantcomponents of a polypeptide's natural environment, e.g. materials thatwould interfere with diagnostic or therapeutic uses for the polypeptidesuch as, for example, enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. A polypeptide can be purified (1) to greaterthan 90%, greater than 95%, or greater than 98% by weight of antibody asdetermined by the Lowry method, for example, more than 99% by weight,(2) to a degree sufficient to obtain at least 15 residues of N-terminalor internal amino acid sequence by use of a spinning cup sequenator, or(3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) under reducing or nonreducing conditionsusing Coomassie blue or silver stain.

As used herein, the term “immune cells” generally includes white bloodcells (leukocytes) which are derived from hematopoietic stem cells (HSC)produced in the bone marrow. “Immune cells” includes, e.g., lymphocytes(T cells, B cells, natural killer (NK) cells) and myeloid-derived cells(neutrophil, eosinophil, basophil, monocyte, macrophage, dendriticcells).

As used herein, “T cell” includes all types of immune cells expressingCD3 including T-helper cells (CD4⁺ cells), cytotoxic T cells (CD8⁺cells). T-regulatory cells (Treg) and gamma-delta T cells.

As used herein, a “cytotoxic cell” includes CD8⁺ T cells, natural-killer(NK) cells, NK-T cells, γδ T cells, a subpopulation of CD4⁺ cells, andneutrophils, which are cells capable of mediating cytotoxicityresponses.

As used herein, the term “stem cell” generally includes pluripotent ormultipotent stem cells. “Stem cells” includes, e.g., embryonic stemcells (ES); mesenchymal stem cells (MSC); induced-pluripotent stem cells(iPS); and committed progenitor cells (hematopoietic stem cells (HSC);bone marrow derived cells, etc.).

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, e.g., in a human, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease.

As used interchangeably herein, the terms “individual”, “subject”,“host”, and “patient” refer to a mammal, including, but not limited to,humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits),non-human primates, humans, canines, felines, ungulates (e.g., equines,bovines, ovines, porcines, caprines), etc.

As used herein, the terms “therapeutically effective amount” or“efficacious amount” refers to the amount of an agent, or combinedamounts of two agents, that, when administered to a mammal or othersubject for treating a disease, is sufficient to affect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the agent(s), the disease and its severity and the age,weight, etc., of the subject to be treated.

As used herein, the term “evolution” or “evolving” refers to using oneor more methods of mutagenesis to generate a different polynucleotideencoding a different polypeptide, which is itself an improved biologicalmolecule and/or contributes to the generation of another improvedbiological molecule. “Physiological” or “normal” or “normalphysiological” conditions are conditions such as, but not limited to,pressure, temperature, pH, ionic strength, osmotic pressure, osmolality,oxidative stress, concentration of one or more solutes, concentration ofelectrolytes, concentration of glucose, concentration of hyaluronan,concentration of lactic acid or lactate, concentration of albumin,levels of adenosine, levels of R-2-hydroxyglutarate, concentration ofpyruvate, concentration of oxygen, and/or presence of oxidants,reductants, or co-factors, as well as other conditions, that would beconsidered within a normal range at the site of administration, or atthe tissue or organ at the site of action, to a subject.

As used herein, a “genetically modified cell” is a cell that contain anexogenous nucleic acid(s) regardless of whether the exogenous nucleicacid(s) is integrated into the genome of the cell. As used herein, a“transduced cell” is a cell that contains an exogenous nucleic acid(s)that is integrated into the genome of the cell.

A “polypeptide” as used herein can include part of or an entire proteinmolecule as well as any posttranslational or other modifications.

A pseudotyping element as used herein can include a “bindingpolypeptide” that includes one or more polypeptides, typicallyglycoproteins, that identify and bind the target host cell, and one ormore “fusogenic polypeptides” that mediate fusion of the retroviral andtarget host cell membranes, thereby allowing a retroviral genome toenter the target host cell. The “binding polypeptide” as used herein,can also be referred to as a “T cell and/or NK cell binding polypeptide”or a “target engagement element,” and the “fusogenic polypeptide” canalso be referred to as a “fusogenic element”.

A “resting” lymphocyte, such as for example, a resting T cell, is alymphocyte in the GO stage of the cell cycle that does not expressactivation markers such as Ki-67. Resting lymphocytes can include naïveT cells that have never encountered specific antigen and memory T cellsthat have been altered by a previous encounter with an antigen. A“resting” lymphocyte can also be referred to as a “quiescent”lymphocyte.

As used herein, “lymphodepletion” involves methods that reduce thenumber of lymphocytes in a subject, for example by administration of alymphodepletion agent. Lymphodepletion can also be attained by partialbody or whole body fractioned radiation therapy. A lymphodepletion agentcan be a chemical compound or composition capable of decreasing thenumber of functional lymphocytes in a mammal when administered to themammal. One example of such an agent is one or more chemotherapeuticagents. Such agents and dosages are known, and can be selected by atreating physician depending on the subject to be treated. Examples oflymphodepletion agents include, but are not limited to, fludarabine,cyclophosphamide, cladribine, denileukin diftitox, or combinationsthereof.

RNA interference (RNAi) is a biological process in which RNA moleculesinhibit gene expression or translation by neutralizing targeted RNAmolecules. The RNA target may be mRNA, or it may be any other RNAsusceptible to functional inhibition by RNAi. As used herein, an“inhibitory RNA molecule” refers to an RNA molecule whose presencewithin a cell results in RNAi and leads to reduced expression of atranscript to which the inhibitory RNA molecule is targeted. Aninhibitory RNA molecule as used herein has a 5′ stem and a 3′ stem thatis capable of forming an RNA duplex. The inhibitory RNA molecule can be,for example, a miRNA (either endogenous or artificial) or a shRNA, aprecursor of a miRNA (i.e. a Pri-miRNA or Pre-miRNA) or shRNA, or adsRNA that is either transcribed or introduced directly as an isolatednucleic acid, to a cell or subject.

As used herein, “double stranded RNA” or “dsRNA” or “RNA duplex” refersto RNA molecules that are comprised of two strands. Double-strandedmolecules include those comprised of two RNA strands that hybridize toform the duplex RNA structure or a single RNA strand that doubles backon itself to form a duplex structure. Most, but not necessarily all ofthe bases in the duplex regions are base-paired. The duplex regioncomprises a sequence complementary to a target RNA. The sequencecomplementary to a target RNA is an antisense sequence, and isfrequently from 18 to 29, from 19 to 29, from 19 to 21, or from 25 to 28nucleotides long, or in some embodiments between 18, 19, 20, 21, 22, 23,24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28 29, or 30 onthe high end, where a given range always has a low end lower than a highend. Such structures typically include a 5′ stem, a loop, and a 3′ stemconnected by a loop which is contiguous with each stem and which is notpart of the duplex. The loop comprises, in certain embodiments, at least3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments the loopcomprises from 2 to 40, from 3 to 40, from 3 to 21, or from 19 to 21nucleotides, or in some embodiments between 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 on the low end and 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 on the high end, wherea given range always has a low end lower than a high end.

The term “microRNA flanking sequence” as used herein refers tonucleotide sequences including microRNA processing elements. MicroRNAprocessing elements are the minimal nucleic acid sequences whichcontribute to the production of mature microRNA from precursor microRNA.Often these elements are located within a 40 nucleotide sequence thatflanks a microRNA stem-loop structure. In some instances the microRNAprocessing elements are found within a stretch of nucleotide sequencesof between 5 and 4,000 nucleotides in length that flank a microRNAstem-loop structure.

The term “linker” when used in reference to a multiplex inhibitory RNAmolecule refers to a connecting means that joins two inhibitory RNAmolecules.

As used herein, a “recombinant retrovirus” refers to a non-replicable,or “replication incompetent”, retrovirus unless it is explicitly notedas a replicable retrovirus. The terms “recombinant retrovirus” and“recombinant retroviral particle” are used interchangeably herein. Suchretrovirus/retroviral particle can be any type of retroviral particleincluding, for example, gamma retrovirus, and in illustrativeembodiments, lentivirus. As is known, such retroviral particles, forexample lentiviral particles, typically are formed in packaging cells bytransfecting the packing cells with plasmids that include packagingcomponents such as Gag, Pol and Rev, an envelope or pseudotyping plasmidthat encodes a pseudotyping element, and a transfer, genomic, orretroviral (e.g. lentiviral) expression vector, which is typically aplasmid on which a gene(s) or other coding sequence of interest isencoded. Accordingly, a retroviral (e.g. lentiviral) expression vectorincludes sequences (e.g. a 5′ LTR and a 3′ LTR flanking e.g. a psipackaging element and a target heterologous coding sequence) thatpromote expression and packaging after transfection into a cell. Theterms “lentivirus” and “lentiviral particle” are used interchangeablyherein.

A “framework” of a miRNA consists of “5′ microRNA flanking sequence”and/or “3′ microRNA flanking sequence” surrounding a miRNA and, in somecases, a loop sequence that separates the stems of a stem-loop structurein a miRNA. In some examples, the “framework” is derived from naturallyoccurring miRNAs, such as, for example, miR-155. The terms “5′ microRNAflanking sequence” and “5′ arm” are used interchangeably herein. Theterms “3′ microRNA flanking sequence” and “3′ arm” are usedinterchangeably herein.

As used herein, the term “miRNA precursor” refers to an RNA molecule ofany length which can be enzymatically processed into an miRNA, such as aprimary RNA transcript, a pri-miRNA, or a pre-miRNA.

As used herein, the term “construct” refers to an isolated polypeptideor an isolated polynucleotide encoding a polypeptide. A polynucleotideconstruct can encode a polypeptide, for example, a lymphoproliferativeelement. A skilled artisan will understand whether a construct refers toan isolated polynucleotide or an isolated polypeptide depending on thecontext.

As used herein, “MOI”, refers to Multiplicity of Infection ratio wherethe MOI is equal to the ratio of the number of virus particles used forinfection per number of cells. Functional titering of the number ofvirus particles can be performed using FACS and reporter expression.

“Peripheral blood mononuclear cells” (PBMCs) include peripheral bloodcells having a round nucleus and include lymphocytes (e.g. T cells, NKcells, and B cells) and monocytes. Some blood cell types that are notPBMCs include red blood cells, platelets and granulocytes (i.e.neutrophils, eosinophils, and basophils).

It is to be understood that the present disclosure and the aspects andembodiments provided herein, are not limited to particular examplesdisclosed, as such may, of course, vary. It is also to be understoodthat tire terminology used herein is for the purpose of disclosingparticular examples and embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Whenmultiple low and multiple high values for ranges are given that overlap,a skilled artisan will recognize that a selected range will include alow value that is less than the high value. All headings in thisspecification are for the convenience of the reader and are notlimiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “achimeric antigen receptor” includes a plurality of such chimeric antigenreceptors and equivalents thereof known to those skilled in the art, andso forth. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

DETAILED DESCRIPTION

The present disclosure overcomes prior art challenges by providingimproved methods and compositions for genetically modifying lymphocytes,for example NK cells and in illustrative embodiments, T cells. Some ofthe methods and compositions herein, provide simplified and more rapidprocesses for transducing lymphocytes that avoid some steps that requirespecialized devices. Furthermore, the methods provide better control ofpost-transduction processing since any such processing is done ex vivo,which therefore allows the option of removing various unwanted cells.Thus, the methods provide an important step toward democratization ofcell therapy methods.

Illustrative methods and compositions for genetically modifyinglymphocytes, for example NK cells and in illustrative embodiments, Tcells, are performed in less time than prior methods. Furthermore,compositions that have many uses, including their use in these improvedmethods, are provided. Some of these compositions are geneticallymodified lymphocytes that have improved proliferative and survivalqualities, including in in vitro culturing, for example in the absenceof growth factors. Such genetically modified lymphocytes will haveutility for example, as research tools to better understand factors thatinfluence T cell proliferation and survival, and for commercialproduction, for example for the production of certain factors, such asgrowth factors and immunomodulatory agents, that can be harvested andtested or used in commercial products.

Methods for Transducing and/or Genetically Modifying Lymphocytes

Provided herein in certain aspects, is a method of transducing and/orgenetically modifying a lymphocyte, such as a (typically a populationof) peripheral blood mononuclear cell (PBMC), typically a T cell and/oran NK cell, and in certain illustrative embodiments a resting T celland/or resting NK cell, that includes contacting the lymphocyte with a(typically a population of) replication incompetent recombinantretroviral particle, wherein the replication incompetent recombinantretroviral particle typically comprises a pseudotyping element on itssurface, wherein said contacting (and incubation under contactingconditions) facilitates membrane association, membrane fusion, andoptionally transduction of the resting T cell and/or NK cell by thereplication incompetent recombinant retroviral particle, therebyproducing the genetically modified T cell and/or NK cell. Inillustrative embodiments, pre-activation of the T cell and/or NK cell isnot required, and an activation element, which can be any activationelement provided herein, is present in a reaction mixture in which thecontacting takes place. In further illustrative embodiments, theactivation element is present on a surface of the replicationincompetent recombinant retroviral particle. In illustrativeembodiments, the activation element is anti-CD3, such as anti-CD3 scFv,or anti-CD3 scFvFc.

In some embodiments, the contacting step and an optional incubationthereafter, which includes a step to remove retroviral particles notassociated with cells, in a method provided herein of transducing and/orgenetically modifying a PBMC or a lymphocyte, typically a T cell and/oran NK cell, can be performed (or can occur), for 72, 48, or 24 hours orless or for any of the contacting time ranges provided herein. However,in illustrative embodiments, the contacting is performed for less than 2hours, less than 1 hour, less than 30 minutes or less than 15 minutes,but in each case there is at least an initial contacting step in whichretroviral particles and cells are brought into contact in suspension ina transduction reaction mixture. This contacting typically includes aninitial step in which retroviral particles that are not associated witha cell of the reaction mixture are separated from the cells, which arethen further processed. Such suspension can include allowing cells andretroviral particles to settle or causing such settling throughapplication of a force, such as a centrifugal force, to the bottom of avessel or chamber, as discussed in further detail herein. Inillustrative embodiments, such g force is lower than the g forces usedsuccessfully in spinoculation procedures. Further contacting times anddiscussions regarding contacting and the optional incubation, arediscussed further herein. In further illustrative embodiments, thecontacting is performed for between an initial contacting step only(without any further incubating in the reaction mixture including theretroviral particles free in suspension and cells in suspension) withoutany further incubation in the reaction mixture, or a 5 minute, 10minute, 15 minute, 30 minute, or 1 hour incubation in the reactionmixture, which can be a step of separating free retroviral particles ina reaction mixture from those associated with cells.

Various embodiments of this method, as well as other aspects, such asuse and NK cells and T cells made by such a method, are disclosed indetail herein. Furthermore, various elements or steps of such methodaspects for transducing and/or genetically modifying a PBMC, lymphocyte,T cell and/or NK cell, are provided herein, for example in this sectionand the Exemplary Embodiments section, and such methods includeembodiments that are provided throughout this specification, as furtherdiscussed herein. For example, embodiments of any of the aspects fortransducing and/or genetically modifying a PBMC or a lymphocyte, forexample an NK cell or in illustrative embodiments, a T cell, providedfor example in this section and in the Exemplary Embodiments section,can include any of the embodiments of replication incompetentrecombinant retroviral particles provided herein, including those thatinclude one or more lymphoproliferative element, CAR, pseudotypingelement, riboswitch, activation element, membrane-bound cytokine, miRNA,Kozak-type sequence, WPRE element, triple stop codon, and/or otherelement disclosed herein, and can be combined with methods herein forproducing retroviral particles using a packaging cell. In certainillustrative embodiments, the retroviral particle is a lentiviralparticle. Such a method for genetically modifying and/or transducing aPBMC or a lymphocyte, such as a T cell and/or NK cell can be performedin vitro or ex vivo. A skilled artisan will recognize that detailsprovided herein for transducing and/or genetically modifying PBMCs orlymphocytes, such as T cell(s) and/or NK cell(s) can apply to any aspectthat includes such step(s).

In certain illustrative embodiments, the cell is genetically modifiedand/or transduced without requiring prior activation or stimulation,whether in vivo, in vitro, or ex vivo. In certain illustrativeembodiments, the cell is activated during the contacting and is notactivated at all or for more than 15 minutes, 30 minutes, 1, 2, 4, or 8hours before the contacting. In certain illustrative embodiments,activation by elements that are not present on the retroviral particlesurface is not required for genetically modifying and/or transducing thecell. Accordingly, such activation or stimulation elements are notrequired other than on the retroviral particle, before, during, or afterthe contacting. Thus, as discussed in more detail herein, theseillustrative embodiments that do not require pre-activation orstimulation provide the ability to rapidly perform in vitro experimentsaimed at better understanding T cells and the biologicals mechanisms,therein. Furthermore, such methods provide for much more efficientcommercial production of biological products produced using PBMCs,lymphocytes, T cells, or NK cells, and development of such commercialproduction methods. Finally, such methods provide for more rapid ex vivoprocessing of PBMCs for adoptive cell therapy, fundamentally simplifyingthe delivery of such therapies, for example by providing point of caremethods.

Compositions and Methods for Transducing Lymphocytes in WholeBloodlymphocytes in Whole Blood

Provided herein in certain aspects, is a method of transducing and/orgenetically modifying peripheral blood mononuclear cells (PBMCs), orlymphocytes, typically T cells and/or NK cells, and in certainillustrative embodiments resting T cells and/or resting NK cells, in areaction mixture comprising blood, or a component thereof, and/or ananticoagulant, that includes contacting the lymphocytes with replicationincompetent recombinant retroviral particles in the reaction mixturethat itself represents a separate aspect provided herein. The reactionmixture in illustrative embodiments comprises the lymphocytes and thereplication incompetent recombinant retroviral particles, a T cellactivation element and one or more additional blood components set outbelow that in illustrative embodiments are present because the reactionmixture comprises at least 10% whole blood, wherein the replicationincompetent recombinant retroviral particles typically comprises apseudotyping element on its surface. In such methods, the contacting(and incubation under contacting conditions) facilitates association ofthe lymphocytes with the replication incompetent recombinant retroviralparticles, wherein the recombinant retroviral particles geneticallymodify and/or transduce the lymphocytes. The reaction mixture of thisaspect comprises at least 10% whole blood (e.g. at least 10%, 20%, 25%,50%, 60%, 70%, 80%, 90%, 95%, or 99% whole blood) and optionally aneffective amount of an anticoagulant, or the reaction mixture furthercomprises at least one additional blood or blood preparation componentthat is not a PBMC, for example the reaction mixture comprises aneffective amount of an anti-coagulant and one or more blood preparationcomponent that is not a PBMC. In illustrative embodiments such blood orblood preparation component that is not a PBMC is one or more (e.g. atleast one, two, three, four, or five) or all of the following additionalcomponents:

-   -   a) erythrocytes, wherein the erythrocytes comprise between 1 and        60% of the volume of the reaction mixture;    -   b) neutrophils, wherein the neutrophils comprise at least 10% of        the white blood cells in the reaction mixture, or wherein the        reaction mixture comprises at least 10% as many neutrophils as T        cells;    -   c) basophils, wherein the basophils comprise at least 0.05% of        the white blood cells in the reaction mixture;    -   d) eosinophils, wherein the reaction mixture comprises at least        0.1% of the white blood cells in the reaction mixture;    -   e) plasma, wherein the plasma comprises at least 1% of the        volume of the reaction mixture; and    -   f) an anti-coagulant    -   (such blood or blood preparation components a-f above referred        to herein as (“Noteworthy Non-PBMC Blood or Blood Preparation        Components”)).

The one or more additional blood components are present in certainillustrative embodiments of the reaction mixture (including related use,genetically modified T cell or NK cell, or method for geneticallymodifying T cells and/or NK cells aspects provided herein) because inthese illustrative embodiments the reaction mixture comprises at least10% whole blood, and in certain illustrative embodiments, at least 25%,50%, 75%, 90%, or 95% whole blood, or for example between 25% and 95%whole blood. In these illustrative embodiments, such reaction mixturesare formed by combining whole blood with an anticoagulant (for exampleby collecting whole blood into a blood collection tube comprising ananti-coagulant), and adding a solution of recombinant retroviruses tothe blood with anticoagulant. Thus, in illustrative embodiments, thereaction mixture comprises an anti-coagulant as set out in more detailherein. In some embodiments, the whole blood is not, or does notcomprise, cord blood.

The reaction mixture in these aspects, typically does not include a PBMCenrichment procedure before the transduction reaction mixture is formed.Thus, typically such reaction mixtures include additional componentslisted in a)-f) above, which are not PBMCs. Furthermore, in illustrativeembodiments, the reaction mixture comprises all of the additionalcomponents listed in a) to e) above, because the reaction mixturecomprises substantially whole blood, or whole blood. “Substantiallywhole blood” is blood that was isolated from an individual(s), has notbeen subjected to a PBMC enrichment procedure, and is diluted by lessthan 50% with other solutions. For example, this dilution can be fromaddition of an anti-coagulant as well as addition of a volume of fluidcomprising retroviral particles. Further reaction mixture embodimentsfor methods and compositions that relate to transducing lymphocytes inwhole blood, are provided herein.

In another aspect, provided herein are genetically modified lymphocytes,in illustrative embodiments genetically modified T cells and/or NK cellsmade by the above method of transducing and/or genetically modifyinglymphocytes in whole blood. In yet another aspect provided herein, isuse of replication incompetent recombinant retroviral particles in themanufacture of a kit for genetically modifying lymphocytes, inillustrative embodiments T cells and/or NK cells of a subject, whereinthe use of the kit comprises the above method of transducing and/orgenetically modifying lymphocytes in whole blood. In another aspect,provided herein are methods for administering genetically modifiedlymphocytes to a subject wherein the genetically modified lymphocytesare produced by the above method of transducing and/or geneticallymodifying lymphocytes in whole blood. Aspects provided herein thatinclude such methods of transducing and/or genetically modifyinglymphocytes in whole blood, uses of such a method in the manufacture ofa kit, reaction mixtures formed in such a method, genetically modifiedlymphocytes made by such a method, and methods for administering agenetically modified lymphocyte made by such a method, are referred toherein as “composition and method aspects for transducing lymphocytes inwhole blood.” It should be noted that although illustrative embodimentsfor such aspects involve contacting T cells and/or NK cells withretroviral particles in whole blood, such aspects also include otherembodiments, where one or more of additional components a-f above, arepresent in transduction reaction mixtures at higher concentrations thanis typical after a PBMC enrichment procedure.

Various elements or steps of such method aspects for transducinglymphocytes in whole blood, are provided herein, for example in thissection and the Exemplary Embodiments section, and such methods includeembodiments that are provided throughout this specification, as furtherdiscussed herein. A skilled artisan will recognize that many embodimentsprovided herein anywhere in this specification can be applied to any ofthe aspects of the composition and method aspects for transducinglymphocytes in whole blood. For example, embodiments of any of thecomposition and method aspects for transducing lymphocytes in wholeblood provided for example in this section and/or in the ExemplaryEmbodiments section, can include any of the embodiments of replicationincompetent recombinant retroviral particles provided herein, includingthose that include one or more polypeptide lymphoproliferative element,inhibitory RNA, CAR, pseudotyping element, riboswitch, activationelement, membrane-bound cytokine, miRNA, Kozak-type sequence, WPREelement, triple stop codon, and/or other element disclosed herein, andcan be combined with methods herein for producing retroviral particlesusing a packaging cell.

As non-limiting examples of embodiments that can be used in many aspectsherein, as discussed in more detail herein, the pseudotyping element istypically capable of binding lymphocytes (e.g. T cells and/or NK cells)in illustrative embodiments resting T cells and/or resting NK cells andfacilitating membrane fusion on its own or in conjunction with otherprotein(s) of the replication incompetent recombinant retroviralparticles. In certain illustrative embodiments, the retroviral particleis a lentiviral particle. Such a method for genetically modifying alymphocyte, such as a T cell and/or NK cell in whole blood, can beperformed in vitro or ex vivo.

Anticoagulants are included in reaction mixtures for certain embodimentsof the composition and method aspects for transducing lymphocytes inwhole blood provided herein. In some illustrative embodiments, blood iscollected with the anti-coagulant present in the collection vessel (e.g.tube or bag), for example using standard blood collection protocolsknown in the art. Anticoagulants that can be used in composition andmethod aspects for transducing lymphocytes in whole blood providedherein include compounds or biologies that block or limit the thrombinblood clotting cascade. The anti-coagulants include: metal chelatingagents, preferably calcium ion chelating agents, such as citrate (e.g.containing free citrate ion), including solutions of citrate thatcontain one or more components such as citric acid, sodium citrate,phosphate, adenine and mono or polysaccharides, for example dextrose,oxalate, and EDTA; heparin and heparin analogues, such as unfractionatedheparin, low molecular weight heparins, and other synthetic saccharides;and vitamin K antagonists such as coumarins. Exemplary citratecompositions include: acid citrate dextrose (ACD) (also calledanticoagulant citrate dextrose solution A and solution B (United StatesPharmacopeia 26, 2002, pp 158)); and a citrate phosphate dextrose (CPD)solution, which can also be prepared as CPD-A1 as is known in the art.Accordingly, the anticoagulant composition may also include phosphateions or monobasic phosphate ion, adenine, and mono or polysaccharides.

Such anti-coagulants can be present in a reaction mixture atconcentrations that are effective for preventing coagulation of blood(i.e. effective amounts) as known in the art, or at a concentration thatis, for example, 2 times, 1.5 times, 1.25 times, 1.2 times, 1.1 times,or 9/10, ⅘, 7/10, ⅗, ½, ⅖, 3/10, ⅕, or 1/10 the effective concentration.The effective concentrations of many different anticoagulants is knownand can be readily determined empirically by analyzing differentconcentrations for their ability to prevent blood coagulation, which canbe physically observed. Numerous coagulometers are availablecommercially that measure coagulation, and various sensor technologiescan be used, for example QCM sensors (See e.g., Yao et al., “BloodCoagulation Testing Smartphone Platform Using Quartz CrystalMicrobalance Dissipation Method,” Sensors (Basel). 2018 September;18(9): 3073). The effective concentration includes the concentration ofany commercially available anti-coagulant in a commercially availabletube or bag after the anti-coagulant is diluted in the volume of bloodintended for the tube or bag. For example, the concentration of acidcitrate dextrose (ACD) in a reaction mixture in certain embodiments ofthe composition and method aspects for transducing lymphocytes in wholeblood provided herein, can be between 0.1 and 5×, or between 0.25 and2.5×, between 0.5 and 2×, between 0.75 and 1.5×, between 0.8 and 1.2×,between 0.9 and 1.1×, about 1×, or 1× the concentration of ACD in acommercially available ACD blood collection tube or bag. For example, ina standard process, blood can be collected into tubes or bags containing3.2% (109 mM) sodium citrate (109 mM) at a ratio of 9 parts blood and 1part anticoagulant. Thus, in certain illustrative embodiments with areaction mixture made by adding 1-2 parts of a retroviral particlesolution to this mixture of 1 part anticoagulant to 9 parts blood, thecitrate concentration can be between for example, 0.25% to 0.4%, or0.30% to 0.35%. In an illustrative standard blood collection embodiment,15 mls of ACD Solution A are present in a blood bag for collecting 100mL of blood. The ACD before addition of blood contains Citric acid(anhydrous) 7.3 g/L (0.73%), Sodium citrate (dihydrate) 22.0 g/L (2.2%),and Dextrose (monohydrate) 24.5 g/L [USP] (2.4%). After addition of 100ml of blood to the bag that contains ACD, a volume of for example,between 5 and 20 mls of the genetically modified retroviral particles isadded. Thus, in some embodiments, the concentration of ACD components ina reaction mixture can be between 0.05 and 0.1%, or 0.06 and 0.08%Citric acid (anhydrous), 0.17 and 0.27, or 0.20 and 0.24 Sodium citrate(dihydrate), 0.2 and 0.3, or 0.20 and 0.28, or 0.22 and 0.26% Dextrose(monohydrate). In certain embodiments, sodium citrate is used at aconcentration of between 0.001 and 0.02 M in the reaction mixture.

In some embodiments, heparin is present in the reaction mixtures, forexample at a concentration between 0.1 and 5×, or between 0.25 and 2.5×,between 0.5 and 2×, between 0.75 and 1.5×, between 0.8 and 1.2×, between0.9 and 1.1×, about 1×, or 1× the concentration of heparin in acommercially available heparin blood collection tube. Heparin is aglycosaminoglycan anticoagulant with a molecular weight ranging from5,000-30,000 daltons. In some embodiments, heparin is used at aconcentration of about 1.5 to 45, 5 to 30, 10 to 20, or 15 USP units/mlof reaction mixture. In some embodiments, the effective concentrationfor EDTA, for example as K₂EDTA, in the reaction mixtures herein can bebetween 0.15 and 5 mg/ml, between 1 and 3 mg/ml between 1.5-2.2 mg/ml ofblood, or between 1 and 2 mg/ml, or about 1.5 mg/ml. The reactionmixtures in composition and method aspects for transducing lymphocytesin whole blood provided herein, can include two or more anticoagulantswhose combined effective dose prevents coagulation of the blood prior toformation of the reaction mixture and/or of the reaction mixture itself.

In some embodiments, the anti-coagulant can be administered to a subjectbefore blood is collected from the subject for ex vivo transduction,such that coagulation of the blood when it is collected in inhibited, atleast partially and at least through a contacting step and optionalincubation period thereafter. In such embodiments, for example acidcitrate dextrose can be administered to the subject at between 80mg/kg/day and 5 mg/kg/day (mg refer to the mg of citric acid and kgapplies to the mammal to be treated). Heparin, can be delivered forexample, at a dose of between 5 units/kg/hr to 30 units/kg/hr.

In addition to, or instead of an anti-coagulant, composition and methodaspects for transducing lymphocytes in whole blood provided herein, caninclude at least one additional component selected from one or more ofthe following components:

-   -   a) erythrocytes, wherein the erythrocytes comprise between 0.1        and 75% of the volume of the reaction mixture;    -   b) neutrophils, wherein the neutrophils comprise at least 10% of        the white blood cells in the reaction mixture, or wherein the        reaction mixture comprises at least 10% as many neutrophils as T        cells;    -   c) basophils, wherein the basophils comprise at least 0.05% of        the white blood cells in the reaction mixture;    -   d) eosinophils, wherein the reaction mixture comprises at least        0.1% of the white blood cells in the reaction mixture;    -   e) plasma, wherein the plasma comprises at least 1% of the        volume of the reaction mixture; and    -   f) platelets, wherein the platelets comprise at least 1×10⁶        platelets/liter of the reaction mixture.

With respect to erythrocytes, in some embodiments, erythrocytes cancomprise between 0.1, 0.5, 1, 5, 10, 25, 35 or 40% of the volume of thereaction mixture on the low end of the range, and between 25, 50, 60, or75% of the volume of the reaction mixture on the high end of the range.In illustrative embodiments, erythrocytes comprise between 1 and 60%,between 10 and 60%, between 20 and 60%, between 30 and 60%, between 40and 60%, between 40 and 50%, between 42 and 48%, between 44 and 46%,about 45% or 45%.

With respect to neutrophils, in some embodiments, neutrophils cancomprise between 0.1, 0.5, 1, 5, 10, 20, 25, 35 or 40% of the whiteblood cells of the reaction mixture on the low end of the range, andbetween 25, 50, 60, 70, 75 and 80% of the white blood cells of thereaction mixture on the high end of the range, for example between 25%and 70%, or between 30% and 60% or between 40% and 60% of the whiteblood cells of the reaction mixture. In some embodiments, moreneutrophils are present than T cells and/or NK cells, in reactionmixtures herein.

With respect to eosinophils in some embodiments, eosinophils cancomprise between 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and1.8% of the white blood cells of the reaction mixture on the low end ofthe range, and between 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.5, 4, 5, 6, 8 and10% of the white blood cells of the reaction mixture on the high end ofthe range. In illustrative embodiments, eosinophils comprise between0.05 and 10.0% between 0.1 and 9%, between 0.2 and 8%, between 0.2 and6%, between 0.5 and 4% between 0.8 and 4%, or between 1 and 4% of thewhite blood cells of the reaction mixture.

With respect to basophils in some embodiments, basophils can comprisebetween 0.05, 0.1, 0.2, 0.4, 0.45 and 0.5% of the white blood cells ofthe reaction mixture on the low end of the range, and between 0.8, 0.9,1.0, 1.1, 1.2, 1.5 and 2.0% of the white blood cells of the reactionmixture on the high end of the range. In illustrative embodiments,basophils comprise between 0.05 and 1.4% between 0.1 and 1.4%, between0.2 and 1.4%, between 0.3 and 1.4%, between 0.4 and 1.4% between 0.5 and1.4% between 0.5 and 1.2%, between 0.5 and 1.1%, or between 0.5 and 1.0%of the white blood cells of the reaction mixture.

With respect to plasma, in some embodiments, plasma can comprise between0.1, 0.5, 1, 5, 10, 25, 35 or 45% of the volume of the reaction mixtureon the low end of the range, and between 25, 50, 60, 70 and 80% of thevolume of the reaction mixture on the high end of the range. Inillustrative embodiments, plasma comprise between 0.1 and 80% between 1and 80% between 5 and 80%, between 10 and 80%, between 30 and 80%,between 40 and 80%, between 45 and 70% between 50 and 60%, between 52and 58% between 54 and 56%, about 55% or 55% of the reaction mixture.

With respect to platelets, in some embodiments, platelets can comprisebetween 1×10⁵, 1×10⁶, 1×10⁷, or 1×10⁸ platelets/mL of the reactionmixture on the low end of the range, and between 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 2×10¹³, or 2×10¹⁴ platelets/mL of the reaction mixture on thehigh end of the range. In illustrative embodiments, platelets comprisebetween 1×10⁵ and 1×10¹² platelets, between 1×10⁶ and 1×10¹¹ platelets,between 1×10⁷ and 1×10¹⁰ platelets, between 1×10⁸, and 1×10⁹platelets/mL, or between 1×10⁸ and 5×10⁸ platelets/ml of the reactionmixture.

Illustrative Cell Processing Methods for Genetically Modifying T Cellsand/or NK Cells in the Presence of Blood, or a Component Thereof

It is noteworthy that some embodiments of methods for geneticallymodifying provided herein do not include a step of collecting blood froma subject. However, as shown in FIG. 1, some of the methods providedherein include a step where blood is collected (110) from a subject.Blood can be collected or obtained from a subject by any suitable methodknown in the art as discussed in more detail herein. For example, theblood can be collected by venipuncture or any other blood collectionmethod by which a sample of blood is collected. In some embodiments, thevolume of blood collected is between 25 ml and 250 ml, for example,between 25 ml and 60 ml, between 50 ml and 90 ml, between 75 ml and 125ml, or between 90 ml and 120 ml, or between 95 and 110 ml.

Regardless of whether blood is collected from a subject, in any of themethod aspects provided herein for genetically modifying lymphocytes(e.g. T cells and/or NK cells), the lymphocytes are contacted withreplication incompetent retroviral particles in a reaction mixture. Inillustrative embodiments, this contacting, and the reaction mixture inwhich the contacting occurs, takes place within a closed cell processingsystem, as discussed in more detail herein. In traditional closed cellprocessing methods that involve genetic modification and/ortransductions of lymphocytes ex vivo, especially in methods forautologous cell therapy, many steps occur over days, such as PBMCenrichment(s), washing(s), cell activation, transduction, expansion,collection, and optionally reintroduction. In more recent methods (SeeFIG. 1A), some of the steps and time involved in this ex vivo cellprocessing have been reduced (See e.g. WO2019/055946). These more recentmethods (as well as the further improved cell processing methodsprovided herein), furthermore use a rapid ex vivo transduction process,for example that includes no or minimal preactivation (e.g. less than30, 15, 10, or 5 minutes of contacting lymphocytes such as T cellsand/or NK cells with an activation agent before they are contacted withretroviral particles). In certain embodiments of such methods, a T celland/or NK cell activation element is present in the reaction mixture inwhich the contacting step occurs. In illustrative embodiments, the Tcell and/or NK cell activation element is associated with surfaces ofretroviral particles present in the reaction mixture. In illustrativeembodiments, such a method is used in a point of care autologous celltherapy method. However, such more recent methods still involve a PBMCenrichment step/procedure (120), which typically takes at least around 1hour within the closed system, followed by cell counting, transfer andmedia addition, which takes at least around 45 additional minutes beforelymphocytes are contacted with retroviral particles to form atransduction reaction mixture (130A). Following the “viral transduction”step, which typically is a contacting step with incubating as discussedin detail herein, lymphocytes are typically washed away from retroviralparticles that remain in suspension (140A), for example using a Sepax,and collected (150A), with the final product typically in an infusionbag for reinfusion or cryopreservation vial for storage (160A). Asdiscussed in further detail herein, traditional PBMC enrichmentprocedures typically involve ficoll density gradients and centrifugal(e.g. centrifugation) or centripetal (e.g. Sepax) forces or useleukophoresis to enrich PBMCs.

As demonstrated in the Examples provided herein, it was surprisinglyfound that lymphocytes (e.g. T cells and/or NK cells) can be contactedwith replication incompetent retroviral particles in a reaction mixtureof whole blood that contains an anti-coagulant, and a significantpercentage of the lymphocytes can be genetically modified andtransduced. Thus, it was discovered that effective genetic modificationof lymphocytes by recombinant retroviral particles can be carried out inthe presence of blood components and blood cells in addition to PBMCs.Furthermore, based on the surprising finding discussed immediately aboveregarding effective genetic modification of T cells and optionally NKcells by retroviral particles even when contacting is performed in wholeblood, provided herein in an illustrative embodiment, is a furthersimplified method in which lymphocytes are genetically modified and/ortransduced by adding replication incompetent retroviral particlesdirectly to whole blood to form a reaction mixture (130B), and cells inthe whole blood are contacted by the replication incompetent retroviralparticles for contacting times with optional incubations providedherein. Such a further improved method in this illustrative embodiment,thus includes no lymphocyte enrichment steps before lymphocytes in wholeblood, typically containing an anti-coagulant, are contacted withretroviral particles. This further improved method, like other cellprocessing methods herein, is typically carried out within a closed cellprocessing system and can include no or minimal preactivation beforelymphocytes are contacted with retroviral particles. In these furthersimplified methods lymphocytes in whole blood can be contacted withretroviral particles directly in a blood bag. After the contacting step(130B) in such methods, lymphocytes that were contacted with retroviralparticles, are washed and concentrated using a PBMC enrichment procedure(135B), which also reduces neutrophils to facilitate reintroduction intoa subject. Thus, in such embodiments, no PBMC enrichment procedure andno lymphocyte-enriching filtration is performed before cells in wholeblood, and typically comprising an anticoagulant, are contacted withrecombinant retroviral particles. However, in the embodiment of FIG. 1B,such a PBMC enrichment method is performed (135B) for example using aSepax with a ficoll gradient, after the contacting with optionalincubation (130B) is carried out. Following the PBMC enrichment,lymphocytes optionally can be washed further away from any retroviralparticles that remain (140B), for example using a Sepax, and collected(150B), with the final product typically in an infusion bag forreinfusion or cryopreservation vial for storage (160B).

FIG. 2 provides a non-limiting illustrative example of a cell processingleukodepletion filtration assembly (200) that enriches nucleated cellsthat can be used as the leukodepletion filter in the methods of FIG. 1.The illustrative leukodepletion filtration assembly (200), which inillustrative embodiments is a single-use filtration assembly, comprisesa leukocyte depletion media (e.g. filter set) within a filter enclosure(210), that has an inlet (225), and an outlet (226), and a configurationof bags, valves and/or channels/tubes that provide the ability toconcentrate, enrich, wash and collect retained white blood cells ornucleated blood cells using perfusion and reverse perfusion (see e.g.EP2602315A1, incorporated by reference herein, in its entirety). In anillustrative embodiment, the leukodepletion filtration assembly (200) isa commercially available HemaTrate filter (Cook Regenetec, Indianapolis,Ind.). Leukodepletion filtration assemblies can be used, to concentratetotal nucleated cells (INC) including granulocytes, which are removed inPBMC enrichment procedures in a closed cell processing system. Since afilter assembly comprising leukocyte depletion media of EP2602315A1 suchas a HemaTrate filter and the illustrative leukodepletion filterassembly of FIG. 2 do not remove granulocytes, they are not consideredPBMC enrichment assemblies or filters herein, and methods thatincorporate them are not considered PBMC enrichment procedures or stepsherein.

The leukodepletion filter assembly (200) of FIG. 2 is a single-usesterile assembly that includes various tubes and valves, typicallyneedle-free valves, that allow isolation of white blood cells from wholeblood and blood cell preparations that include leukocytes, as well asrapid washing and concentrating of white blood cells. In thisillustrative assembly, a blood bag (215), for example a 500 ml PVC bagcontaining about 120 ml of a transduction/contacting reaction mixturecomprising whole blood, an anti-coagulant, and retroviral particles isconnected to the assembly (200) at a first assembly opening (217) of aninlet tubing (255), after the reaction mixture is subjected to acontacting step with optional incubation, as disclosed in detail herein.Lymphocytes, including some T cells and/or NK cells with associatedretroviral particles, and some that could be genetically modified atthis point, as well as other blood cells and components in the wholeblood reaction mixture as well as the anti-coagulant enter the inlettubing (255) through the first assembly opening (217) by gravitationalforce when a clamp on the first inlet tubing (255) is released. Thegenetically modified T cells and/or NK cells pass through a inlet valve(247) and a collection valve (245), to enter a filter enclosure (210)through a filter enclosure inlet (225) to contact a leukodepletion IVfilter set (e.g. SKU J1472A Jorgensen Labs) within the filter enclosure(210). Nucleated blood cells including leukocytes are retained by thefilter, but other blood components pass through the filter and out thefilter enclosure outlet (226) into the outlet tubing (256), then throughan outlet valve (247) and are collected in a waste collection bag (216),which for example can be a 2 L PVC waste collection bag.

An optional buffer wash step can be performed by switching inlet valve(247) to a wash position. In this optional wash step, a buffer bag(219), for example a 500 ml saline wash bag, is connected to a secondassembly opening (218) of inlet tubing (255). The buffer moves into theinlet tubing (255) through the second assembly opening (218) bygravitational force when a clamp on the inlet tubing (255) is released.The buffer passes through inlet valve (247) and collection valve (245),to enter filter enclosure (210) through the filter enclosure inlet (225)and passes through the leukodepletion filter set within the filterenclosure (210) to rinse the lymphocytes retained on the filter. Thebuffer moves out the filter enclosure outlet (226) into the outlettubing (256), then through an outlet valve (247) and is collected in awaste collection bag (216), which can be the same waste collection bagas used to collect reaction mixture components that passed through thefilter in the previous step, or a new waste collection bag swapped inplace of the first waste collection bag before the buffer was allowed toenter the second assembly opening (218). The optional wash step can beoptionally performed multiple times by repeating the above process withadditional buffer.

Once the entire or substantially the entire volume of the reactionmixture in the blood bag (215) passes over the filter (210), and theoptional washing step(s) is optionally performed, a reverse perfusionprocess is initiated to move fluid in an opposite direction in theassembly (200) to collect lymphocytes retained on the filter set withinthe filter enclosure (210). Illustrative embodiments of leukodepletionfilter assemblies herein are adaptable for reperfusion. Beforeinitiating the reverse perfusion process in the illustrative assembly(200), the outlet valve (247) is switched to a reperfusion position andthe collection valve (245) is switched to a collection position. Toinitiate reperfusion, a buffer (e.g. PBS) in syringe (266), which forexample can be a 25 ml syringe, is passed into outlet tubing (256) byinjection using syringe (266). The buffer then enters the filterenclosure (210) through the filter enclosure outlet (226) and moveslymphocytes retained on the filter set out of the filter enclosure (210)through the filter enclosure inlet (225) and into the inlet tubing(255). Then lymphocytes, including some T cells and/or NK cells withassociated retroviral particles, some of which could be geneticallymodified and/or transduced at this point, are collected in a cell samplecollection bag (265), which for example can be a 25 ml cryopreservationbag, after the pass through the collection valve (245).

In some aspects, provided herein is a kit for genetically modifying NKcells and/or in illustrative embodiments, T cells. The kit includes aleukodepletion filtration assembly and any of the replicationincompetent retroviral vector embodiments disclosed herein, typicallycontained in a tube or vial. The leukodepletion filtration assembly insuch a kit typically includes a leukodepletion filter or aleukodepletion filter set, typically within a filter enclosure, asexemplified by the illustrative assembly of FIG. 2, as well as aplurality of connected sterile tubes and a plurality of valves connectedthereto, that are adapted for use in a single-use closed bloodprocessing system. Such a kit optionally includes a blood collectionbag, in illustrative embodiments comprising an anti-coagulant, a bloodprocessing buffer bag, a blood processing waste collection bag, a bloodprocessing cell sample collection bag, and a sterile syringe. Inillustrative embodiments, the kit includes a T cell activation elementas disclosed in detail herein, for example anti-CD3. Such activationelement can be provided in solution in the tube or vial containing theretroviral particle, or in a separate tube or vial. In illustrativeembodiments, the activation element is an anti-CD3 associated with asurface of the replication incompetent retroviral particle. Inillustrative embodiments, the replication incompetent recombinantretroviral particles in the kit comprise a polynucleotide comprising oneor more transcriptional units operatively linked to a promoter active inT cells and/or NK cells, wherein the one or more transcriptional unitsencode a first polypeptide comprising a chimeric antigen receptor (CAR)and optionally a lymphoproliferative element, according to any of theembodiments provided herein.

Steps and Reaction Mixtures for Methods for Genetically ModifyingLymphocytes

Some embodiments of any methods used in any aspects provided herein,which are typically methods for genetically modifying lymphocytes,PBMCs, and in illustrative embodiments NK cells and/or in furtherillustrative embodiments, T cells, can include a step of collectingblood from a subject. The blood includes blood components includingblood cells such as lymphocytes (e.g. T cells and NK cells) that can beused in methods and compositions provided herein. In certainillustrative embodiments, the subject is a human subject afflicted withcancer (i.e. a human cancer subject). It is noteworthy that certainembodiments, do not include such a step. However, in embodiments thatinclude collecting blood from a subject, blood can be collected orobtained from a subject by any suitable method known in the art asdiscussed in more detail herein. For example, the blood can be collectedby venipuncture or any other blood collection method by which a sampleof blood is collected. In some embodiments, the volume of bloodcollected is between 50 ml and 250 ml, for example, between 75 ml and125 ml, or between 90 ml and 120 ml, or between 95 and 110 ml. In someembodiments, the volume of blood collected can be between 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or900 ml on the low end of the range and 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225,250, 275, 300, 350, 400, 450, 500, 600, 700, 800, or 900 ml or 1 L onthe high end of the range. In some embodiments, lymphocytes (e.g. Tcells and/or NK cells) can be obtained by apheresis. In someembodiments, the volume of blood taken and processed during apheresiscan be between 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.25, or 1.5 totalblood volumes of a subject on the low end of the range and 0.6, 0.7,0.75, 0.8, 0.9, 1, 1.25, 1.5 1.75, 2, 2.25, or 2.5 total blood volumesof a subject on the high end of the range. The total blood volume of ahuman typically ranges from 4.5 to 6 L and thus much more blood is takenand processed during apheresis than if the blood is collected and thenlymphocytes therein are genetically modified and/or transduced, as inillustrative embodiments herein.

Regardless of whether blood is collected from a subject, in any of themethod aspects provided herein for genetically modifying lymphocytes(e.g. T cells and/or NK cells), the lymphocytes are contacted withreplication incompetent retroviral particles in a reaction mixture. Thecontacting in any embodiment provided herein, can be performed forexample in a chamber of a closed system adapted for processing of bloodcells, for example within a blood bag, as discussed in more detailherein. The transduction reaction mixture can include one or morebuffers, ions, and a culture media. With respect to retroviralparticles, and in illustrative embodiments, lentiviral particles, incertain exemplary reaction mixtures provided herein, between 0.1 and 50,0.5 and 50, 0.5 and 20, 0.5 and 10, 1 and 25, 1 and 15, 1 and 10, 1 and5, 2 and 15, 2 and 10, 2 and 7, 2 and 3, 3 and 10, 3 and 15, or 5 and15, multiplicity of infection (MOI); or at least 1 and less than 6, 11,or 51 MOI; or in some embodiments, between 5 and 10 MOI units ofreplication incompetent recombinant retroviral particles are present. Insome embodiments, the MOI can be at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10or 15. With respect to composition and method for transducinglymphocytes in blood, in certain embodiments higher MOI can be used thanin methods wherein PBMCs are isolated and used in the reaction mixtures.For example, illustrative embodiments of compositions and methods fortransducing lymphocytes in whole blood, assuming 1×10⁶ PBMCs/ml ofblood, can use retroviral particles with an MOI of between 1 and 50, 2and 25, 2.5 and 20, 2.5 and 10, 4 and 6, or about 5, and in someembodiments between 5 and 20, 5 and 15, 10 and 20, or 10 and 15.

In illustrative embodiments, this contacting, and the reaction mixturein which the contacting occurs, takes place within a closed cellprocessing system, as discussed in more detail herein. A packaging cell,and in illustrative embodiments a packaging cell line, and inparticularly illustrative embodiments a packaging cell provided incertain aspects herein, can be used to produce the replicationincompetent recombinant retroviral particles. The lymphocytes in thereaction mixture can be PBMCs, or in aspects herein that providecompositions and methods for transducing lymphocytes in whole blood, ananti-coagulant and/or an additional blood component, includingadditional types of blood cells that are not PBMCs, as discussed herein.In fact, in illustrative embodiments of these composition and methodaspects for transducing lymphocytes in whole blood, the reaction mixturecan essentially be whole blood, and typically an anti-coagulant,retroviral particles, and a small amount of the solution in which theretroviral particles were delivered to the whole blood.

In some reaction mixture provided herein, T-cells can be present forexample, between 10, 20, 30, or 40% of the lymphocytes of the reactionmixture on the low end of the range, and between 40, 50, 60, 70, 80, or90% of the lymphocytes of the reaction mixture on the high end of therange. In illustrative embodiments, T-cells comprise between 10 and 90%,between 20 and 90%, between 30 and 90%, between 40 and 90%, between 40and 80%, between 45% to 75% or of the lymphocytes. In such embodiments,for example, NK cells can be present at between 1, 2, 3, 4, or 5% of thelymphocytes of the reaction mixture on the low end of the range, andbetween 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14% of the lymphocytes of thereaction mixture on the high end of the range. In illustrativeembodiments, T-cells comprise between 1 and 14%, between 2 and 14%,between 3 and 14%, between 4 and 14%, between 5 and 14%, between 5 to13%, between 5 to 12%, between 5 to 11% or, between 5 to 10% of thelymphocytes of the reaction mixture.

In reaction mixtures that relate to composition and method aspects forgenetically modifying lymphocytes in whole blood provided herein,lymphocytes, including NK cells and T cells, can be present at a lowerpercent of blood cells, and at a lower percentage of white blood cells,in the reaction mixture than methods that involve a PBMC enrichmentprocedure before forming the reaction mixture. For example, in someembodiments of these aspects, more granulocytes or neutrophils arepresent in the reaction mixture than NK cells or even T cells. Detailsregarding compositions of anti-coagulants and one or more additionalblood components present in the reaction mixtures of aspects forgenetically modifying lymphocytes in whole blood, are provided in detailin other sections herein.

As disclosed herein, composition and method aspects for transducinglymphocytes in whole blood typically do not involve a PBMC enrichmentstep of a blood sample, before lymphocytes from the blood sample arecontacted with retroviral particles in the reaction mixtures disclosedherein for those aspects. However, in some embodiments,neutrophils/granulocytes are separated away from other blood cellsbefore the cells are contacted with replication incompetent recombinantretroviral particles. In some embodiments, peripheral blood mononuclearcells (PBMCs) including peripheral blood lymphocytes (PBLs) such as Tcell and/or NK cells, are isolated away from other components of a bloodsample using for example, a PBMC enrichment procedure, before they arecombined into a reaction mixture with retroviral particles.

A PBMC enrichment procedure is a procedure in which PBMCs are enrichedat least 25-fold, and typically at least 50-fold from other blood celltypes. For example, it is believed that PBMCs make up less than 1% ofblood cells in whole blood. After a PBMC enrichment procedure, at least30%, and in some examples as many as 70% of cells isolated in the PBMCfraction are PBMCs. It is possible that even higher enrichment of PBMCsis achieved using some PBMC enrichment procedures. Various differentPBMC enrichment procedures are known in the art. For example, a PBMCenrichment procedure is a ficoll density gradient centrifugation processthat separates the main cell populations, such as lymphocytes,monocytes, granulocytes, and red blood cells, throughout a densitygradient medium. In such a method the aqueous medium includes ficoll, ahydrophilic polysaccharide that forms the high density solution.Layering of whole blood over or under a density medium without mixing ofthe two layers followed by centrifugation will disperse the cellsaccording to their densities with the PBMC fraction forming a thin whitelayer at the interface between the plasma and the density gradientmedium (see e.g. Panda and Ravindran (2013) Isolation of Human PBMCs.BioProtoc. Vol. 3(3)). Furthermore, centripetal forces can be used toseparate PBMCs from other blood components, in ficoll using the spinningforce of a Sepax cell processing system.

In another PBMC enrichment method, an automated leukapheresis collectionsystem (such as SPECTRA OPTIA® APHERESIS SYSTEM form TERUMO BCT, INC.Lakewood Colo. 80215, USA) is used to separate the inflow of whole bloodfrom the target PBMC fraction using high-speed centrifugation whiletypically returning the outflow material, such as plasma, red bloodcells, and granulocytes, back to the donor, although this returningwould be optional in methods provided herein. Further processing may benecessary to remove residual red blood cells and granulocytes. Bothmethods include a time intensive purification of the PBMCs, and theleukapheresis method requires the presence and participation of thepatient during the PBMC enrichment step.

As further non-limiting examples of PBMC enrichment procedures, in someembodiments for methods of transducing or genetically modifying herein,PBMCs are isolated using a Sepax or Sepax 2 cell processing system(BioSafe). In some embodiments, the PBMCs are isolated using a CliniMACSProdigy cell processor (Miltenyi Biotec). In some embodiments, anautomated apheresis separator is used which takes blood from thesubject, passes the blood through an apparatus that sorts out aparticular cell type (such as, for example, PBMCs), and returns theremainder back into the subject. Density gradient centrifugation can beperformed after apheresis. In some embodiments, the PBMCs are isolatedusing a leukodepletion filter assembly. In some embodiments, magneticbead activated cell sorting is then used for purifying a specific cellpopulation from PBMCs, such as, for example, PBLs or a subset thereof,according to a cellular phenotype (i.e. positive selection), before theyare used in a reaction mixture herein.

Other methods for purification can also be used, such as, for example,substrate adhesion, which utilizes a substrate that mimics theenvironment that a T cell encounters during recruitment, to purify Tcells before adding them to a reaction mixture, or negative selectioncan be used, in which unwanted cells are targeted for removal withantibody complexes that target the unwanted cells for removal before areaction mixture for a contacting step is formed. In some embodiments,red blood cell resetting can be used to remove red blood cells beforeforming a reaction mixture. In other embodiments, hematopoietic stemcells can be removed before a contacting step, and thus in theseembodiments, are not present during the contacting step. In someembodiments herein, especially for compositions and methods fortransducing lymphocytes in whole blood, an ABC transporter inhibitorand/or substrate is not present before, during, or both before andduring the contacting (i.e. not present in the reaction mixture in whichcontacting takes place) with or without optional incubating, or any stepof the method.

In certain illustrative embodiments for any aspects provided herein,lymphocytes are genetically modified and/or transduced without prioractivation or stimulation, and/or without requiring prior activation orstimulation, whether in vivo, in vitro, or ex-vivo; and/or furthermore,in some embodiments, without ex vivo or in vitro activation orstimulation after an initial contacting with or without an optionalincubation, or without requiring ex vivo or in vitro activation orstimulation after an initial contacting with or without an optionalincubation. Thus, in illustrative embodiments, some, most, at least 25%,50%, 60%, 70%, 75%, 80%, 90%, at least 95%, at least 99% or all of thelymphocytes are resting when they are combined with retroviral particlesto form a reaction mixture, and typically are resting when they arecontacted with retroviral viral particles in a reaction mixture. Inmethods for genetically modifying lymphocytes such as T cells and/or NKcells in blood or a component thereof, lymphocytes can be contacted inthe typically resting state they were in when present in the collectedblood in vivo immediately before collection. In some embodiments, the Tcells and/or NK cells consist of between 95 and 100% resting cells(Ki-67). In some embodiments, the T cell and/or NK cells that arecontacted by replication incompetent recombinant retroviral particlesinclude between 90, 91, 92, 93, 94, and 95% resting cells on the low endof the range and 96, 97, 98, 99, or 100% resting cells on the high endof the range. In some embodiments, the T cells and/or NK cells includenaïve cells. In some illustrative embodiments, the subembodiments inthis paragraph are included in composition and method aspects fortransducing lymphocytes in whole blood.

Contact between the T cells and/or NK cells and the replicationincompetent recombinant retroviral particles can facilitate transductionof the T cells and/or NK cells by the replication incompetentrecombinant retroviral particles. Not to be limited by theory, duringthe period of contact, the replication incompetent recombinantretroviral particles identify and bind to T cells and/or NK cells atwhich point the retroviral and host cell membranes start to fuse. Then,as a next step in the process of transduction, genetic material from thereplication incompetent recombinant retroviral particles enters the Tcells and/or NK cells at which time the T cells and/or NK cells are“genetically modified” as the phrase is used herein. It is noteworthythat such process might occur hours or even days after the contacting isinitiated, and even after non-associated retroviral particles are rinsedaway. Then the genetic material is typically integrated into the genomicDNA of the T cells and/or NK cells, at which time the T cells and/or NKcells are now “transduced” as the term is used herein. Accordingly, inillustrative embodiments, any method for genetically modifyinglymphocytes (e.g. T cells and/or NK cells) herein, is a method fortransducing lymphocytes (e.g. T cells and/or NK cells). It is believedthat by day 6 in vivo or ex vivo, after contacting is initiated, thevast majority of genetically modified cells have been transduced.Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505. Throughout this disclosure, a transduced T cell and/orNK cell includes progeny of ex vivo transduced cells that retain atleast some of the nucleic acids or polynucleotides that are incorporatedinto the genome of a cell during the ex vivo transduction. In methodsherein that recite “reintroducing” a transduced cell, it will beunderstood that such cell is typically not in a transduced state when itis collected from the blood of a subject.

Many of the methods provided herein include genetic modification andtransduction of T cells and/or NK cells. Methods are known in the artfor genetically modifying and transducing T cells and/or NK cells exvivo with replication incompetent recombinant retroviral particles, suchas replication incompetent recombinant lentiviral particles. Methodsprovided herein, in illustrative embodiments, do not require ex vivostimulation or activation. Thus, this common step in prior methods canbe avoided in the present method, although ex vivo stimulatorymolecule(s) such as anti-CD3 and/or anti-CD28 beads, can be presentduring the contacting and optional incubation thereafter. However, withillustrative methods provided herein, ex vivo stimulation is notrequired.

In certain illustrative embodiments for any aspects herein, the bloodcells, such as lymphocytes, and especially T cells and/or NK cells areactivated during the contacting or an optional incubation thereafter,and are not activated at all or for more than 15 minutes, 30 minutes, 1,2, 4, or 8 hours before the contacting. In certain illustrativeembodiments, activation by elements that are not present on theretroviral particle surface is not required for genetically modifyingthe lymphocytes. Accordingly, such activation or stimulation elementsare not required other than on tire retroviral particle, before, during,or after the contacting. Thus, as discussed in more detail herein, theseillustrative embodiments that do not require pre-activation orstimulation provide the ability to rapidly perform in vitro experimentsaimed at better understanding T cells and the biologicals mechanisms,therein. Furthermore, such methods provide for much more efficientcommercial production of biological products produced using PBMCs,lymphocytes, T cells, or NK cells, and development of such commercialproduction methods. Finally, such methods provide for more rapid ex vivoprocessing of lymphocytes (e.g. NK cells and especially T cells) foradoptive cell therapy, fundamentally simplifying the delivery of suchtherapies, for example by providing point of care methods.

Although in illustrative embodiments, T cells and/or NK cells are notactivated prior to being contacted with a recombinant retrovirus inmethods herein, a T cell activation element in illustrative embodimentsis present in the reaction mixture where initial contacting of arecombinant retrovirus and lymphocytes occurs. For example, such T cellactivation element can be in solution in tire reaction mixture. Forexample, soluble anti-CD3 antibodies can be present in the reactionmixture during the contacting and optional incubation thereafter, at25-200, 50-150, 75-125, or 100 ng/ml. In illustrative embodiments, the Tcell activation element is associated with the retroviral surface. The Tcell activation element can be any T cell activation element providedherein. In illustrative embodiments, the T cell activation element canbe anti-CD3, such as anti-CD3 scFv, or anti-CD3 scFvFc. Accordingly, insome embodiments, the replication incompetent recombinant retroviralparticle can further include a T cell activation element, which infurther illustrative examples is associated with the external side ofthe surface of the retrovirus.

The contacting step of a method for transducing and/or a method forgenetically modifying lymphocytes in whole blood, provided herein,typically includes an initial step in which the retroviral particle,typically a population of retroviral particles, are brought into contactwith blood cells, typically a population of blood cells that includes ananti-coagulant and/or additional blood components other than PBMCs, thatare not present after a PBMC enrichment procedure, while in suspensionin a liquid buffer and/or media to form a transduction reaction mixture.This contacting, as in other aspects provided herein, can be followed byan optional incubating period in this reaction mixture that includes theretroviral particles and the blood cells comprising lymphocytes (e.g. Tcells and/or NK cells) in suspension. In methods for geneticallymodifying T cells and/or NK cells in blood or a component thereof, thereaction mixture can include at least one, two, three, four, five, orall additional blood components as disclosed herein, and in illustrativeembodiments includes one or more anticoagulants.

The transduction reaction mixture in any of the aspects provided hereincan be incubated at between 23 and 39° C., and in some illustrativeembodiments at 37° C., in an optional incubation step after the initialcontacting of retroviral particles and lymphocytes. In certainembodiments, the transduction reaction can be carried out at 37-39° C.for faster fusion/transduction. The cells and retroviral particles whenbrought into contact in the transduction reaction mixture can beimmediately processed to remove the retroviral particles that remainfree in suspension and not associated with cells, from the cells.Optionally, the cells in suspension and retroviral particles whetherfree in suspension or associated with the cells in suspension, can beincubated for various lengths of time, as provided herein for acontacting step in a method provided herein. Before further steps, awash can be performed, regardless of whether such cells will be studiedin vitro, ex vivo or introduced into a subject.

Illustrative methods are disclosed herein for genetically modifyinglymphocytes, especially NK cells and in illustrative embodiments, Tcells, that are much shorter and simpler than prior methods.Accordingly, in some embodiments, the contacting step in any methodprovided herein of transducing and/or genetically modifying a PBMC or alymphocyte, typically a T cell and/or an NK cell, can be performed (orcan occur) for any of the time periods provided in this specification,including, but not limited to those provided in the ExemplaryEmbodiments section. For example, said contacting can be for less than24 hours, for example, less than 12 hours, less than 8 hours, less than4 hours, less than 2 hours, less than 1 hour, less than 30 minutes orless than 15 minutes, but in each case there is at least an initialcontacting step in which retroviral particles and cells come intocontact in suspension in a transduction reaction mixture beforeretroviral particles that remain in suspension not associated with acell, are separated from cells and typically discarded, as discussed infurther detail herein. It should be noted, but not intending to belimited by theory, that it is believed that contacting begins at thetime that retroviral particles and lymphocytes are combined together,typically by adding a solution containing the retroviral particles intoa solution containing lymphocytes (e.g. T cells and/or NK cells).

After such initial contacting, in some embodiments there is anincubating of the reaction mixture containing cells and retroviralparticles in suspension for a specified time period without removingretroviral particles that remain free in solution and not associatedwith cells. This incubating is sometimes referred to herein as anoptional incubation. Thus, In illustrative embodiments, the contacting(including initial contacting and optional incubation) can be performed(or can occur) (where as indicated in general herein the low end of aselected range is less than the high end of the selected range) forbetween 30 seconds or 1, 2, 5, 10, 15, 30 or 45 minutes, or 1, 2, 3, 4,5, 6, 7, or 8 hours on the low end of the range, and between 10 minutes,15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, and 72hours on the high end of the range. In certain illustrative embodiments,the contacting step can be performed for between 30 seconds, 1 minute, 5minutes, 10 minutes, 15 minutes, or 30 minutes on the low end of therange and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12hours on the high end of the range. In some embodiments, the contactingstep is performed for between 30 seconds, 1 minute, and 5 minutes on thelow end of the range, and 10 minutes, 15 minutes, 30 minutes, 1 hour, 2hours, 4 hours, or 8 hours on the high end of the range. Thus, in someembodiments, after the time when a reaction mixture is formed by addingretroviral particles to lymphocytes, the reaction mixture can beincubated for between 5 minutes and 12 hours, between 5 minutes and 10hours, between 5 minutes and 8 hours, between 5 minutes and 6 hours,between 5 minutes and 4 hours, between 5 minutes and 2 hours, between 5minutes and 1 hour, between 5 minutes and 30 minutes, or between 5minutes and 15 minutes. In other embodiments, the reaction mixture canbe incubated for between 15 minutes and 12 hours, between 15 minutes and10 hours, between 15 minutes and 8 hours, between 15 minutes and 6hours, between 15 minutes and 4 hours, between 15 minutes and 2 hours,between 15 minutes and 1 hour, between 15 minutes and 45 minutes, orbetween 15 minutes and 30 minutes. In other embodiments, the reactionmixture can be incubated for between 30 minutes and 12 hours, between 30minutes and 10 hours, between 30 minutes and 8 hours, between 30 minutesand 6 hours, between 30 minutes and 4 hours, between 30 minutes and 2hours, between 30 minutes and 1 hour, between 30 minutes and 45 minutes.In other embodiments, the reaction mixture can be incubated for between1 hour and 12 hours, between 1 hour and 8 hours, between 1 hour and 4hours, or between 1 hour and 2 hours. In another illustrativeembodiment, the contacting is performed for between an initialcontacting step only (without any further incubating in the reactionmixture including the retroviral particles free in suspension and cellsin suspension) without any further incubation in the reaction mixture,or a 5 minute, 10 minute, 15 minute, 30 minute, or 1 hour incubation inthe reaction mixture.

After the indicated time period fertile initial contacting and optionalincubation that can be part of the contacting step, blood cells or a Tcell and/or NK cell-containing fraction thereof in the reaction mixture,are separated from retroviral particles that are not associated withsuch cells. For example, this can be performed using a PBMC enrichmentprocedure (e.g. a Ficoll gradient in a Sepax unit), or in certainillustrative embodiments provided herein, by filtering the reactionmixture over a leukocyte depletion filter set assembly, and thencollecting the leukocytes, which include T cells and NK cells. Inanother embodiment, this can be performed by centrifugation of thereaction mixture at a relative centrifugal force less than 500 g, forexample 400 g, or between 300 and 490 g, or between 350 and 450 g. Suchcentrifugation to separate retroviral particles from cells can beperformed for example, for between 5 minutes and 15 minutes, or between5 minutes and 10 minutes. In illustrative embodiments where centrifugalforce is used to separate cells from retroviral particles that are notassociated with cells, such g force is typically lower than the g forcesused successfully in spinoculation procedures.

In some illustrative embodiments, a method provided herein in anyaspect, does not involve performing a spinoculation. In someembodiments, spinoculation is included as part of a contacting step. Inillustrative embodiments, when spinoculation is performed there is noadditional incubating as part of the contacting, as the time of thespinoculation provides the incubation time of the optional incubationdiscussed above. In other embodiments, there is an additional incubationafter the spinoculating of between 15 minutes and 4 hours, or between 15minutes and 2 hours, or between 15 minutes and 1 hour. The spinoculationcan be performed for example, for 30 minutes to 120 minutes, typicallyfor at least 60 minutes, for example for 60 minutes to 180 minutes, or60 minutes to 90 minutes. The spinoculation is typically performed in acentrifuge with a relative centrifugal force of at least 800 g, and moretypically at least 1,200 g, for example between 800 g and 2400 g, orbetween 800 g and 1800 g, or between 1200 g and 2400 g, or between 1200g and 1800 g. After the spinoculation, such methods typically involve anadditional step of resuspending the pelleted cells and retroviralparticles, and then removing retroviral particles that are notassociated with cells according to steps discussed above whenspinoculation is not performed.

The contacting step including the optional incubation therein, and thespinoculation, in embodiments that include spinoculation, can beperformed at between 4 C and 42 C, or between 20 C and 37 C. In certainillustrative embodiments, spinoculation is not performed and thecontacting and associated optional incubation are carried out at 20-25Cfor 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes orless, 15 minutes or less, or 15 minutes to 2 hours, 15 minutes to 1hour, or 15 minutes to 30 minutes.

In some embodiments of the methods and compositions disclosed herein,between 5% and 85% of the total lymphocytes collected from the blood aregenetically modified. In some embodiments, the percent of lymphocytesthat are genetically modified and/or transduced is between 1, 5, and 10%on the low end of the range, and 15, 20, 25, 30, 40, 50, 60, 70, 80, and85% on the high end of the range. In some embodiments, the percent of Tcells and NK cells that are genetically modified and/or transduced is atleast 5%, at least 10%, at least 15%, or at least 20%. As illustrated intire Examples herein, in exemplary methods provided herein fortransducing lymphocytes in whole blood, between 1% and 20%, or between1% and 15%, or between 5% and 15%, or between 7% and 12% or about 10% oflymphocytes are genetically modified and/or transduced.

Methods of genetically modifying lymphocytes provided according to anymethod herein, typically include insertion into the cell, of apolynucleotide comprising one or more transcriptional units encoding aCAR or a lymphoproliferative element, or in illustrative embodimentsencoding both a CAR and a lymphoproliferative element according to anyof the CAR and lymphoproliferative element embodiments provided herein.Such CAR and lymphoproliferative elements can be provided to support theshorter and more simplified methods provided herein, which can supportexpansion of genetically modified and/or transduced T cells and/or NKcells after the contacting and optional incubation. Accordingly, inexemplary embodiments of any methods provided herein,lymphoproliferative elements can be delivered from the genome of theretroviral particles inside genetically modified and/or transduced Tcells and/or NK cells, such that those cells have the characteristics ofincreased proliferation and/or survival disclosed in theLymphoproliferative Elements section herein. In exemplary embodiments ofany methods provided herein, the genetically modified T cell or NK cellis capable of engraftment in vivo in mice and/or enrichment in vivo inmice for at least 7, 14, or 28 days. A skilled artisan will recognizethat such mice may be treated or otherwise genetically modified so thatany immunological differences between the genetically modified T celland/or NK cell do not result in an immune response being elicited in themice against any component of the lymphocyte transduced by thereplication incompetent recombinant retroviral particle.

Media that can be included in a contacting step, for example when thecells and retroviral particles are initially brought into contact, or inany aspects provided herein, during optional incubation periods with thereaction mixture thereafter that include retroviral particles and cellsin suspension in the media, or media that can be used during cellculturing and/or during various wash steps in any aspects providedherein, can include base media such as commercially available media forex vivo T cell and/or NK cell culture. Non-limiting examples of suchmedia include, X-VIVO™ 15 Chemically Defined, Serum-free HematopoieticCell Medium (Lonza) (2018 catalog numbers BE02-060F, BE02-00Q,BE-02-061Q, 04-744Q, or 04-418Q), ImmunoCult™-XF T Cell Expansion Medium(STEMCELL Technologies) (2018 catalog number 10981), PRIME-XV® T CellExpansion XSFM (Irvine Scientific) (2018 catalog number 91141), AIM V®Medium CTS™ (Therapeutic Grade) (Thermo Fisher Scientific (Referred toherein as “Thermo Fisher”), or CTS™ Optimizer™ media (Thermo Fisher)(2018 catalog numbers A10221-01 (basal media (bottle)), and A10484-02(supplement), A10221-03 (basal media (bag)), A1048501 (basal media andsupplement kit (bottle)) and, A1048503 (basal media and supplement kit(bag)). Such media can be a chemically defined, serum-free formulationmanufactured in compliance with cGMP. The media can be xeno-free andcomplete. In some embodiments, the base media has been cleared byregulatory agencies for use in ex vivo cell processing, such as an FDA510(k) cleared device. In some embodiments, the media is the basal mediawith or without the supplied T cell expansion supplement of 2018 catalognumber A1048501 (CTS™ OpTmizer™ T Cell Expansion SFM, bottle format) orA1048503 (CTS™ OpTmizer™ T Cell Expansion SFM, bag format) bothavailable from Thermo Fisher (Waltham, Mass.). Additives such as humanserum albumin, human AB+ serum, and/or serum derived from the subjectcan be added to the transduction reaction mixture. Supportive cytokinescan be added to the transduction reaction mixture, such as IL2, IL7, orIL15, or those found in human sera. dGTP can be added to thetransduction reaction in certain embodiments.

In some embodiments of any method herein that includes a step ofgenetically modifying lymphocytes (e.g. T cells and/or NK cells), thecells can be contacted with a retroviral particle without prioractivation. In some embodiments of any method herein that includes astep of genetically modifying T cells and/or NK cells, the T cellsand/or NK cells have not been incubated on a substrate that adheres tomonocytes for more than 4 hours in one embodiment, or for more than 6,hours in another embodiment, or for more than 8 hours in anotherembodiment before the transduction. In one illustrative embodiment, theT cells and/or NK cells have been incubated overnight on an adherentsubstrate to remove monocytes before the transduction. In anotherembodiment, the method can include incubating the T cells and/or NKcells on an adherent substrate that binds monocytes for no more than 30minutes, 1 hour, or 2 hours before the transduction. In anotherembodiment, the T cells and/or NK cells are exposed to no step ofremoving monocytes by an incubation on an adherent substrate before saidtransduction step. In another embodiment, the T cells and/or NK cellsare not incubated with or exposed to a bovine serum, such as a cellculturing bovine serum, for example fetal bovine serum before or duringa contacting step and/or a genetically modifying and/or transductionstep.

Some or all of the steps of the methods for genetically modifyingprovided herein, or uses of such methods, are performed in a closedsystem. Thus, reaction mixtures formed in such methods, and geneticallymodified and/or transduced lymphocytes (e.g. T cells and/or NK cells)made by such methods, can be contained within such a closed system. Aclosed system is a cell processing system that is generally closed orfully closed to an environment, such as an environment within a room oreven the environment within a hood, outside of the conduits such astubes, and chambers, of the system in which cells are processed and/ortransported. One of the greatest risks to safety and regulatory controlin the cell processing procedure is the risk of contamination throughfrequent exposure to the environment as is found in traditional opencell culture systems. To mitigate this risk, particularly in the absenceof antibiotics, some commercial processes have been developed that focuson the use of disposable (single-use) equipment. However, even withtheir use under aseptic conditions, there is always a risk ofcontamination from the opening of flasks to sample or add additionalgrowth media. To overcome this problem, methods provided herein, whichare typically ex vivo methods, are typically performed within aclosed-system. Such a process is designed and can be operated such thatthe product is not exposed to the outside environment. Material transferoccurs via sterile connections, such as sterile tubing and sterilewelded connections. Air for gas exchange can occur via a gas permeablemembrane, via 0.2 μm filter to prevent environmental exposure. In someillustrative embodiments, the methods are performed on T cells, forexample to provide genetically modified T cells.

Such closed system methods can be performed with commercially availabledevices. Different closed system devices can be used at different stepswithin a method and the cells can be transferred between these devicesusing tubing and connections such as welded, luer, spike, or clave portsto prevent exposure of the cells or media to the environment. Forexample, blood can be collected into an IV bag or syringe, optionallyincluding an anti-coagulant, and transferred to a Sepax 2 device(Biosafe) for PBMC enrichment and isolation. In other embodiments, wholeblood can be filtered to collect leukocytes using a leukodepletionfilter assembly. The isolated PBMCs or isolated leukocytes can betransferred to a chamber of a G-Rex device for an optional activation, atransduction and optional expansion. Alternatively, collected blood canbe transduced in a blood bag, for example, the bag in which it wascollected. Finally, the cells can be harvested and collected intoanother bag using a Sepax 2 device. The methods can be carried out inany device or combination of devices adapted for closed system T celland/or NK cell production. Non-limiting examples of such devices includeG-Rex devices (Wilson Wolf), GatheRex (Wilson Wolf), Sepax 2 (Biosafe),WAVE Bioreactors (General Electric), a CultiLife Cell Culture bag(Takara), a PermaLife bag (OriGen), CliniMACS Prodigy (Miltenyi Biotec),and VueLife bags (Saint-Gobain). In illustrative embodiments, theoptional activating, the transducing and optional expanding can beperformed in the same chamber or vessel in the closed system. Forexample, in illustrative embodiments, the chamber can be a chamber of aG-Rex device and PBMCs or leukocytes can be transferred to the chamberof the G-Rex device after they are enriched and isolated, and can remainin the same chamber of the G-Rex device until harvesting.

Methods provided herein can include transferring blood and cells thereinand/or fractions thereof, as well as lymphocytes before or after theyare contacted with retroviral particles, between vessels within a closedsystem, which thus is without environmental exposure. Vessels used inthe closed system, for example, can be a tube, bag, syringe, or othercontainer. In some embodiments, the vessel is a vessel that is used in aresearch facility. In some embodiments, the vessel is a vessel used incommercial production. In other embodiments, the vessel can be acollection vessel used in a blood collection process. Methods forgenetically modifying herein, typically involve a contacting stepwherein lymphocytes are contacted with a replication incompetentrecombinant retroviral particle. The contacting in some embodiments, canbe performed in the vessel, for example, within a blood bag. Blood andvarious lymphocyte-containing fractions thereof, can be transferred fromthe vessel to another vessel (for example from a first vessel to asecond vessel) within the closed system for the contacting. The secondvessel can be a cell processing compartment of a closed device, such asa G-Rex device. In some embodiments, after the contacting thegenetically modified (e.g. transduced) cells can be transferred to adifferent vessel within the closed system (i.e. without exposure to theenvironment). Either before or after this transfer the cells aretypically washed within the closed system to remove substantially all orall of the retroviral particles. In some embodiments, a processdisclosed herein, from collection of blood, to contacting (e.g.transduction), optional incubating, and post-incubation isolation andoptional washing, is performed for between 15 minutes, 30 minutes, or 1,2, 3, or 4 hours on the low end of the range, and 4, 8, 10, or 12 hourson the high end of the range.

Not to be limited by theory, in non-limiting illustrative methods, thedelivery of a polynucleotide encoding a lymphoproliferative element, toa resting T cell and/or NK cell ex vivo, which can integrate into thegenome of the T cell or NK cell, provides that cell with a driver for invivo expansion without the need for lymphodepleting the host. Thus, inillustrative embodiments, the subject is not exposed to alymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28days, or within 1 month, 2 months, 3 months or 6 months of performingthe contacting, during the contacting, and/or within 1, 2, 3, 4, 5, 6,7, 10, 14, 21, or 28 days, or within 1 month, 2 months, 3 months or 6months after the modified T cells and/or NK cells are reintroduced backinto the subject. Furthermore, in non-limiting illustrative embodiments,methods provided herein can be performed without exposing the subject toa lymphodepleting agent during a step wherein a replication incompetentrecombinant retroviral particle is in contact with resting T cellsand/or testing NK cells of the subject and/or during the entire ex vivomethod. Hence, methods of expanding genetically modified T cells and/orNK cells in a subject in vivo is a feature of some embodiments of thepresent disclosure. In illustrative embodiments, such methods are exvivo propagation-free or substantially propagation-free.

This entire method/process from blood draw from a subject toreintroduction of blood back into the subject after ex vivo transductionof T cells and/or NK cells, in non-limiting illustrative embodiments ofany aspects provided herein, can occur over a time period less than 48hours, less than 36 hours, less than 24 hours, less than 12 hours, lessthan 11 hours, less than 10 hours, less titan 9 hours, less than 8hours, less than 7 hours, less than 6 hours, less than 5 hours, lessthan 4 hours, less than 3 hours, 2 hours, or less than 2 hours. In otherembodiments, the entire method/process from blood draw/collection from asubject to reintroduction of blood back into the subject after ex vivotransduction of T cells and/or NK cells, in non-limiting illustrativeembodiments herein, occurs over a time period between 1 hour and 12hours, or between 2 hours and 8 hours, or between 1 hour and 3 hours, orbetween 2 hours and 4 hours, or between 2 hours and 6 hours, or between4 hours and 12 hours, or between 4 hours and 24 hours, or between 8hours and 24 hours, or between 8 hours and 36 hours, or between 8 hoursand 48 hours, or between 12 hours and 24 hours, or between 12 hours and36 hours, or between 12 hours and 48 hours, or over a time periodbetween 15, 30, 60, 90, 120, 180, and 240 minutes on the low end of therange, and 120, 180, and 240, 300, 360, 420, and 480 minutes on the highend of the range. In other embodiments, the entire method/process fromblood draw/collection from a subject to reintroduction of blood backinto the subject after ex vivo transduction of T cells and/or NK cells,occurs over a time period between 1, 2, 3, 4, 6, 8, 10, and 12 hours onthe low end of the range, and 8, 9, 10, 11, 12, 18, 24, 36, or 48 hourson the high end of the range. In some embodiments, the geneticallymodified T cells and/or NK cells are separated from the replicationincompetent recombinant retroviral particles after the time period inwhich contact occurs.

Because methods provided herein for genetically modifying lymphocytes,and associated methods for performing adoptive cell therapy can beperformed in significantly less time than prior methods, fundamentalimprovements in patient care and safety as well as productmanufacturability are made possible. Therefore, such processes areexpected to be favorable in the view of regulatory agencies responsiblefor approving such processes when carried out in vivo for therapeuticpurposes. For example, the subject in non-limiting examples of anyaspects provided herein that include a subject, can remain in the samebuilding (e.g. infusion clinic) or room as the instrument processingtheir blood or sample for the entire time that the sample is beingprocessed before modified T cells and/or NK cells are reintroduced intothe patient. In non-limiting illustrative embodiments, a subject remainswithin line of site and/or within 100, 50, 25, or 12 feet or arm'sdistance of their blood or cells that are being processed, for theentire method/process from blood draw/collection from the subject toreintroduction of blood to the subject after ex vivo transduction of Tcells and/or NK cells. In other non-limiting illustrative embodiments, asubject remains awake and/or at least one person can continue to monitorthe blood or cells of the subject that are being processed, throughoutand/or continuously for the entire method/process from blooddraw/collection from the subject to reintroduction of blood to thesubject after ex vivo transduction of T cells and/or NK cells. Becauseof improvements provided herein, the entire method/process for adoptivecell therapy and/or for transducing resting T cells and/or NK cells fromblood draw/collection from the subject to reintroduction of blood to thesubject after ex vivo transduction of T cells and/or NK cells can beperformed with continuous monitoring by a human. In other non-limitingillustrative embodiments, at no point during the entire method/processfrom blood draw/collection from the subject to reintroduction of bloodto the subject after ex vivo transduction of T cells and/or NK cells,are blood cells incubated in a room that does not have a person present.In other non-limiting illustrative embodiments, the entiremethod/process from blood draw/collection from the subject toreintroduction of blood to the subject after ex vivo transduction of Tcells and/or NK cells, is performed next to the subject and/or in thesame room as the subject and/or next to the bed or chair of the subject.Thus, sample identity mix-ups can be avoided, as well as long andexpensive incubations over periods of days or weeks. This is furtherprovided by the fact that methods provided herein are readily adaptableto closed and automated blood processing systems, where a blood sampleand its components that will be reintroduced into the subject, only makecontact with disposable, single-use components.

Methods for genetically modifying and/or transducing lymphocytes such asT cells and/or NK cells provided herein, can be part of a method forperforming adoptive cell therapy. Typically, methods for performingadoptive cell therapy include steps of collecting blood from a subject,and returning genetically modified and/or transduced lymphocytes (e g Tcells and/or NK cells) to the subject. The present disclosure providesvarious treatment methods using a CAR. A CAR of the present disclosure,when present in a T lymphocyte or an NK cell, can mediate cytotoxicitytoward a target cell. A CAR of the present disclosure binds to anantigen present on a target cell, thereby mediating killing of a targetcell by a T lymphocyte or an NK cell genetically modified to produce theCAR. The ASTR of the CAR binds to an antigen present on the surface of atarget cell. The present disclosure provides methods of killing, orinhibiting the growth of, a target cell, the method involving contactinga cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NKcell) that is genetically modified to produce a subject CAR, such thatthe T lymphocyte or NK cell recognizes an antigen present on the surfaceof a target cell, and mediates killing of the target cell. The targetcell can be a cancer cell, for example, and autologous cell therapymethods herein, can be methods for treating cancer, in some illustrativeembodiments. In these embodiments, the subject can be a an animal orhuman suspected of having cancer, or more typically, a subject that isknown to have cancer.

In some embodiments of any of the methods provided herein forgenetically modifying lymphocytes (e.g. T cells and/or NK cells), andaspects directed to use of replication incompetent recombinantretroviral particles in the manufacture of a kit for geneticallymodifying T cells and/or NK cells of a subject, the genetically modifiedand/or transduced lymphocyte (e.g. T cell and/or NK cell) or populationthereof, are introduced or reintroduced into the subject. Introductionor reintroduction of the genetically modified lymphocytes into a subjectcan be via any route known in the art. For example, introduction orreintroduction can be delivery via infusion into a blood vessel of thesubject. In some embodiments, the genetically modified and/or transducedlymphocyte (e.g. T cell and/or NK cell) or population thereof, undergo 4or fewer cell divisions ex vivo prior to being introduced orreintroduced into the subject. In some embodiments, the lymphocyte(s)used in such a method are resting T cells and/or resting NK cells thatare in contact with the replication incompetent recombinant retroviralparticles for between 1 hour and 12 hours. In some embodiments, no morethan 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hourpass(es) between the time blood is collected from the subject and thetime the genetically modified T cells and/or NK cells are reintroducedinto the subject. In some embodiments, all steps after the blood iscollected and before the blood is reintroduced, are performed in aclosed system in which a person monitors the closed system throughoutthe processing.

In some embodiments of the methods and compositions disclosed herein,the genetically modified T cells and/or NK cells are introduced back,reintroduced, reinfused or otherwise delivered into the subject withoutadditional ex vivo manipulation, such as stimulation and/or activationof T cells and/or NKs. In the prior art methods, ex vivo manipulation isused for stimulation/activation of T cells and/or NK cells and forexpansion of genetically modified T cells and/or NK cells prior tointroducing the genetically modified T cells and/or NK cells into thesubject. In prior art methods, this generally takes days or weeks andrequires a subject to return to a clinic for a blood infusion days orweeks after an initial blood draw. In some embodiments of the methodsand compositions disclosed herein, T cells and/or NK cells are notstimulated ex vivo by exposure to anti-CD3/anti-CD28 solid supports suchas, for example, beads coated with anti-CD3/anti-CD28, prior tocontacting the T cells and/or NK cells with the replication incompetentrecombinant retroviral particles. As such provided herein is an ex vivopropagation-free method. In other embodiments, genetically modified Tcells and/or NK cells are not expanded ex vivo, or only expanded for asmall number of cell divisions (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10rounds of cell division), but are rather expanded, or predominantlyexpanded, in vivo, i.e. within the subject. In some embodiments, noadditional media is added to allow for further expansion of the cells.In some embodiments, no cell manufacturing of the primary bloodlymphocytes (PBLs) occurs while the PBLs are contacted with thereplication incompetent recombinant retroviral particles. Inillustrative embodiments, no cell manufacturing of the PBLs occurs whilethe PBLs are ex vivo. In traditional methods of adoptive cell therapy,subjects are lymphodepleted prior to reinfusion with geneticallymodified T cells and or NK cells. In some embodiments, patients orsubjects are not lymphodepleted prior to blood being withdrawn. In someembodiments, patients or subjects are not lymphodepleted prior toreinfusion with genetically modified T cells and or NK cells. However,the embodiments of the methods and compositions disclosed herein can beused on pre-activated or pre-stimulated T cells and/or NK cells as well.In some embodiments, T cells and/or NK cells can be stimulated ex vivoby exposure to anti-CD3/anti-CD28 solid supports prior to contacting theT cells and/or NK cells with the replication incompetent recombinantretroviral particles. In some embodiments, the T cells and/or NK cellscan be exposed to anti-CD3/anti-CD28 solid supports for less than 1, 2,3, 4, 6, 8, 10, 12, 14, 16, 18, or 24 hours, including no exposure,before the T cells and/or NK cells are contacted the replicationincompetent recombinant retroviral particles. In illustrativeembodiments, the T cells and/or NK cells can be exposed toanti-CD3/anti-CD28 solid supports for less than 1, 2, 3, 4, 6, or 8hours before the T cells and/or NK cells are contacted the replicationincompetent recombinant retroviral particles.

In some illustrative embodiments, cells are introduced or reintroducedinto the subject by infusion into a vein or artery. In any of theembodiments disclosed herein, the number of T cells and/or NK cells tobe reinfused into a subject can be between 1×10³, 2.5×10³, 5×10³, 1×10⁴,2.5×10⁴, 5×10⁴, 1×10⁵, 2.5×10⁵, 5×10⁵, 1×10⁶, 2.5×10⁶, 5×10⁶, and 1×10⁷cells/kg on the low end of the range and 5×10⁴, 1×10⁵, 2.5×10⁵, 5×10⁵,1×10⁶, 2.5×10⁶, 5×10⁶, 1×10⁷, 2.5×10⁷, 5×10⁷, and 1×10⁸ cells/kg on thehigh end of the range. In illustrative embodiments, the number of Tcells and/or NK cells to be reinfused or otherwise delivered into asubject can be between 1×10⁴, 2.5×10⁴, 5×10⁴, and 1×10⁵ cells/kg on thelow end of the range and 2.5×10⁴, 5×10⁴, 1×10⁵, 2.5×10⁵, 5×10⁵ and 1×10⁶cells/kg on the high end of the range. In some embodiments, the numberof PBLs to be reinfused or otherwise delivered into a subject can befewer than 5×10⁵, 1×10⁶, 2.5×10⁶, 5×10⁶, 1×10⁷, 2.5×10⁷, 5×10⁷, and1×10⁸ cells and the low end of the range and 2.5×10⁶, 5×10⁶, 1×10⁷,2.5×10⁷, 5×10⁷, 1×10⁸, 2.5×10⁸, 5×10⁸, and 1×10⁹ cells on the high endof the range. In some embodiments, the number of T cells and/or NK cellsavailable for infusion or reinfusion into a 70 kg subject or patient isbetween 7×10⁵ and 2.5×10⁸ cells. In other embodiments, the number of Tcells and/or NK cells available for transduction is approximately 7×10⁶plus or minus 10%.

Engineered Signaling Polypeptide(S)

In some embodiments, the replication incompetent recombinant retroviralparticles used to contact T cells and/or NK cells have a polynucleotideor nucleic acid having one or more transcriptional units that encode oneor more engineered signaling polypeptides. In some embodiments, anengineered signaling polypeptide includes any combination of anextracellular domain (e.g. an antigen-specific targeting region orASTR), a stalk and a transmembrane domain, combined with one or moteintracellular activating domains, optionally one or more modulatorydomains (such as a co-stimulatory domain), and optionally one or mote Tcell survival motifs. In illustrative embodiments, at least one, two, orall of the engineered signaling polypeptides is a chimeric antigenreceptor (CAR) or a lymphoproliferative element (LE) such as a chimericlymphoproliferative element (CLE). In some embodiments, at least one,two, or all of the engineered signaling polypeptides is a recombinant Tcell receptor (TCR). In some embodiments, when two signalingpolypeptides are utilized, one encodes a lymphoproliferative element andthe other encodes a chimeric antigen receptor (CAR) that includes anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain. For any domain of an engineeredsignaling polypeptide disclosed herein, exemplary sequences can be foundin WO2019/055946, incorporated herein in its entirety by reference. Askilled artisan will recognize that such engineered polypeptides canalso be referred to as recombinant polypeptides. The engineeredsignaling polypeptides, such as CARs, recombinant TCRs, LEs, and CLEsprovided herein, are typically transgenes with respect to lymphocytes,especially T cells and NK cells, and most especially T cells and/or NKcells that are engineered using methods and compositions providedherein, to express such signaling polypeptides.

Extracellular Domain

In some embodiments, an engineered signaling polypeptide includes anextracellular domain that is a member of a specific binding pair. Forexample, in some embodiments, the extracellular domain can be theextracellular domain of a cytokine receptor, or a mutant thereof, or ahormone receptor, or a mutant thereof. Such mutant extracellular domainsin some embodiments have been reported to be constitutively active whenexpressed at least in some cell types. In illustrative embodiments, suchextracellular and transmembrane domains do not include a ligand bindingregion. It is believed that such domains do not bind a ligand whenpresent in an engineered signaling polypeptide and expressed in B cells,T cells, and/or NK cells. Mutations in such receptor mutants can occurin the extracellular juxtamembrane region. Not to be limited by theory,a mutation in at least some extracellular domains (and someextracellular-transmembrane domains) of engineered signalingpolypeptides provided herein, are responsible for signaling of theengineered signaling polypeptide in the absence of ligand, by bringingactivating chains together that are not normally together. Furtherembodiments regarding extracellular domains that comprise mutations inextracellular domains can be found, for example, in theLymphoproliferative Element section herein.

In certain illustrative embodiments, the extracellular domain comprisesa dimerizing motif. In an illustrative embodiment the dimerizing motifcomprises a leucine zipper. In some embodiments, the leucine zipper isfrom a jun polypeptide, for example c-jun. Further embodiments regardingextracellular domains that comprise a dimerizing motif can be found, forexample, in the Lymphoproliferative Element section herein.

In certain embodiments, the extracellular domain is an antigen-specifictargeting region (ASTR), sometimes called an antigen binding domainherein. Specific binding pairs include, but are not limited to,antigen-antibody binding pairs; ligand-receptor binding pairs; and thelike. Thus, a member of a specific binding pair suitable for use in anengineered signaling polypeptide of the present disclosure includes anASTR that is an antibody, an antigen, a ligand, a receptor bindingdomain of a ligand, a receptor, a ligand binding domain of a receptor,and an affibody.

An ASTR suitable for use in an engineered signaling polypeptide of thepresent disclosure can be any antigen-binding polypeptide. In certainembodiments, the ASTR is an antibody such as a full-length antibody, asingle-chain antibody, an Fab fragment, an Fab′ fragment, an (Fab′)₂fragment, an Fv fragment, and a divalent single-chain antibody or adiabody.

In some embodiments, the ASTR is a single chain Fv (scFv). In someembodiments, the heavy chain is positioned N-terminal of the light chainin the engineered signaling polypeptide. In other embodiments, the lightchain is positioned N-terminal of the heavy chain in the engineeredsignaling polypeptide. In any of the disclosed embodiments, the heavyand light chains can be separated by a linker as discussed in moredetail herein. In any of the disclosed embodiments, the heavy or lightchain can be at the N-terminus of the engineered signaling polypeptideand is typically C-terminal of another domain, such as a signal sequenceor peptide.

Other antibody-based recognition domains (cAb VHH (camelid antibodyvariable domains) and humanized versions, IgNAR VH (shark antibodyvariable domains) and humanized versions, sdAb VH (single domainantibody variable domains) and “camelized” antibody variable domains aresuitable for use with the engineered signaling polypeptides and methodsusing the engineered signaling polypeptides of the present disclosure.In some instances, T cell receptor (TCR) based recognition domains.

Certain embodiments for any aspect or embodiment herein that includes aCAR, include CARs having extracellular domains engineered to co-opt theendogenous TCR signaling complex and CD3Z signaling pathway. In oneembodiment, a chimeric antigen receptor ASTR is fused to one of theendogenous TCR complex chains (e.g. TCR alpha, CD3E etc) to promoteincorporation into the TCR complex and signaling through the endogenousCD3Z chains. In other embodiments, a CAR contains a first scFv orprotein that binds to the TCR complex and a second scFv or protein thatbinds to the target antigen (e.g. tumor antigen). In another embodimentthe TCR can be a single chain TCR (scTv, single chain two-domain TCRcontaining VαVβ). Finally, scFv's may also be generated to recognize thespecific MHC/peptide complex, thereby acting as a surrogate TCR. Suchpeptide/MHC scFv-binders may be used in many similar configurations asCAR's.

In some embodiments, the ASTR can be multispecific, e.g. bispecificantibodies. Multispecific antibodies have binding specificities for atleast two different sites. In certain embodiments, one of the bindingspecificities is for one target antigen and the other is for anothertarget antigen. In certain embodiments, bispecific antibodies may bindto two different epitopes of a target antigen. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express atarget antigen. Bispecific antibodies can be prepared as full-lengthantibodies or antibody fragments.

An ASTR suitable for use in an engineered signaling polypeptide of thepresent disclosure can have a variety of antigen-binding specificities.In some cases, the antigen-binding domain is specific for an epitopepresent in an antigen that is expressed by (synthesized by) a targetcell. In one example, the target cell is a cancer cell associatedantigen. The cancer cell associated antigen can be an antigen associatedwith, e.g., a breast cancer cell, a B cell lymphoma, a Hodgkin lymphomacell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, alung cancer cell (e.g., a small cell lung cancer cell), a non-HodgkinB-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancercell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lungcancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, anacute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, aglioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancercell associated antigen may also be expressed by a non-cancerous cell.

Non-limiting examples of antigens to which an ASTR of an engineeredsignaling polypeptide can bind include, e.g., CD19, CD20, CD38, CD30,ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA),epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelialgrowth factor receptor-2 (VEGFR2), high molecular weight-melanomaassociated antigen (HMW-MAA), MAGE-Al, IL-13R-a2, GD2, Axl, Ror2, andthe like.

In some embodiments, a member of a specific binding pair suitable foruse in an engineered signaling polypeptide is an ASTR that is a ligandfor a receptor. Ligands include, but are not limited to, hormones (e.g.erythropoietin, growth hormone, leptin, etc.); cytokines (e.g.,interferons, interleukins, certain hormones, etc.); growth factors(e.g., heregulin; vascular endothelial growth factor (VEGF); and thelike); an integrin-binding peptide (e.g., a peptide comprising thesequence Arg-Gly-Asp (SEQ ID NO:1); and the like.

Where the member of a specific binding pair in an engineered signalingpolypeptide is a ligand, the engineered signaling polypeptide can beactivated in the presence of a second member of the specific bindingpair, where the second member of the specific binding pair is a receptorfor the ligand. For example, where the ligand is VEGF, the second memberof the specific binding pair can be a VEGF receptor, including a solubleVEGF receptor.

As noted above, in some cases, the member of a specific binding pairthat is included in an engineered signaling polypeptide is an ASTR thatis a receptor, e.g., a receptor for a ligand, a co-receptor, etc. Thereceptor can be a ligand-binding fragment of a receptor. Suitablereceptors include, but are not limited to, a growth factor receptor(e.g., a VEGF receptor); a killer cell lectin-like receptor subfamily K,member 1 (NKG2D) polypeptide (receptor for MICA, MICE, and ULB6); acytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.);CD27; a natural cytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337)polypeptide (receptor for HLA-B-associated transcript 3 (BAT3) andB7-H6); etc.); etc.

In certain embodiments of any of the aspects provided herein thatinclude an ASTR, the ASTR can be directed to an intermediate proteinthat links the ASTR with a target molecule expressed on a target cell.The intermediate protein may be endogenously expressed or introducedexogenously and may be natural, engineered, or chemically modified. Incertain embodiments the ASTR can be an anti-tag ASTR such that at leastone tagged intermediate, typically an antibody-tag conjugate, isincluded between a tag recognized by the ASTR and a target molecule,typically a protein target, expressed on a target cell. Accordingly, insuch embodiments, the ASTR binds a tag and the tag is conjugated to anantibody directed against an antigen on a target cell, such as a cancercell. Non-limiting examples of tags include fluorescein isothiocyanate(FITC), streptavidin, biotin, histidine, dinitrophenol, peridininchlorophyll protein complex, green fluorescent protein, phycoerythrin(PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalinephosphatase, glucose oxidase, and maltose binding protein. As such, theASTR comprises a molecule that binds the tag.

Stalk

In some embodiments, the engineered signaling polypeptide includes astalk which is located in the portion of the engineered signalingpolypeptide lying outside the cell and interposed between the ASTR andthe transmembrane domain. In some embodiments, the stalk has at least85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-type CD8 stalkregion (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (SEQ ID NO:2), hasat least 85, 90, 95, 96, 97, 98, 99, or 100% identity to a wild-typeCD28 stalk region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ IDNO:3), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to awild-type immunoglobulin heavy chain stalk region. In an engineeredsignaling polypeptide, the stalk employed allows the antigen-specifictargeting region, and typically the entire engineered signalingpolypeptide, to retain increased binding to a target antigen.

The stalk region can have a length of from about 4 amino acids to about50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aato about 15 aa, from about 15 aa to about 20 aa, from about 20 aa toabout 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about40 aa, or from about 40 aa to about 50 aa.

In some embodiments, the stalk of an engineered signaling polypeptideincludes at least one cysteine. For example, In some embodiments, thestalk can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO:4). Ifpresent, a cysteine in the stalk of a first engineered signalingpolypeptide can be available to form a disulfide bond with a stalk in asecond engineered signaling polypeptide.

Stalks can include immunoglobulin hinge region amino acid sequences thatare known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad.Sci. USA 87:162; and Buck et al. (1986) Nucl. Acids Res. 14:1779. Asnon-limiting examples, an immunoglobulin hinge region can include adomain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or100% sequence identity to a stretch of at least 10, 15, 20, or all ofthe amino acids of any of the following amino acid sequences: DKTHT (SEQID NO:5); CPPC (SEQ ID NO:4); CPEPKSCDTPPPCPR (SEQ ID NO:6) (see, e.g.,Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ IDNO:7); KSCDKTHTCP (SEQ ID NO:8); KCCVDCP (SEQ ID NO:9); KYGPPCP (SEQ IDNO:10); EPKSCDKTHTCPPCP (SEQ ID NO:11) (human IgGl hinge); ERKCCVECPPCP(SEQ ID NO:12) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:13)(human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:14) (human IgG4 hinge); andthe like. The stalk can include a hinge region with an amino acidsequence of a human IgG1, IgG2, IgG3, or IgG4, hinge region. The stalkcan include one or more amino acid substitutions and/or insertionsand/or deletions compared to a wild-type (naturally-occurring) hingeregion. For example, His229 of human IgG 1 hinge can be substituted withTyr, so that the stalk includes the sequence EPKSCDKTYTCPPCP (SEQ IDNO:15), (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891). Thestalk can include an amino acid sequence derived from human CD8; e.g.,the stalk can include the amino acid sequence:TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:16), or avariant thereof.

Transmembrane Domain

An engineered signaling polypeptide of the present disclosure caninclude transmembrane domains for insertion into a eukaryotic cellmembrane. The transmembrane domain can be interposed between the ASTRand the co-stimulatory domain. The transmembrane domain can beinterposed between the stalk and the co-stimulatory domain, such thatthe chimeric antigen receptor includes, in order from the amino terminus(N-terminus) to the carboxyl terminus (C-terminus): an ASTR; a stalk; atransmembrane domain; and an activating domain.

Any transmembrane (TM) domain that provides for insertion of apolypeptide into the cell membrane of a eukaryotic (e.g., mammalian)cell is suitable for use in aspects and embodiments disclosed herein.

Non-limiting examples of TM domains suitable for any of the aspects orembodiments provided herein, include a domain with at least 50, 60, 70,75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to astretch of at least 10, 15, 20, or all of the amino acids of any of thefollowing TM domains or combined stalk and TM domains: a) CD8 alpha TM(SEQ ID NO:17); b) CD8 beta TM (SEQ ID NO:18); c) CD4 stalk (SEQ IDNO:19); d) CD3Z TM (SEQ ID NO:20); e) CD28 TM (SEQ ID NO:21); f) CD134(OX40) TM: (SEQ ID NO:22); g) CD7 TM (SEQ ID NO:23); h) CD8 stalk and TM(SEQ ID NO:24); and i) CD28 stalk and TM (SEQ ID NO:25).

As non-limiting examples, a transmembrane domain of an aspect of theinvention can have at least 80%, 90%, or 95% or can have 100% sequenceidentity to the SEQ ID NO:17 transmembrane domain, or can have 100%sequence identity to any of the transmembrane domains from the followinggenes respectively: the CD8 beta transmembrane domain, the CD4transmembrane domain, the CD3 zeta transmembrane domain, the CD28transmembrane domain, the CD134 transmembrane domain, or the CD7transmembrane domain.

Intracellular Activating Domain

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure when activated,typically induce the production of one or more cytokines; increase celldeath; and/or increase proliferation of CD8⁺ T cells, CD4⁺ T cells, NKTcells, γδ T cells, and/or neutrophils. Activating domains can also bereferred to as activation domains herein. Activating domains can be usedin CARs or in lymphoproliferative elements provided herein.

In some embodiments, the intracellular activating domain includes atleast one (e.g., one, two, three, four, five, six, etc.) ITAM motifs asdescribed below. In some embodiments, an intracellular activating domainof an aspect of the invention can have at least 80%, 90%, or 95% or canhave 100% sequence identity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B,DAP12, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains as describedbelow.

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure include immunoreceptortyrosine-based activation motif (ITAM)-containing intracellularsignaling polypeptides. An ITAM motif is YX₁X₂L/I, where X₁ and X₂ areindependently any amino acid. In some embodiments, the intracellularactivating domain of an engineered signaling polypeptide includes 1, 2,3, 4, or 5 ITAM motifs. In some embodiments, an ITAM motif is repeatedtwice in an intracellular activating domain, where the first and secondinstances of the ITAM motif are separated from one another by 6 to 8amino acids, e.g., (YX₁X₂L/I)(X₃)_(n)(YX₁X₂L/I), where n is an integerfrom 6 to 8, and each of the 6-8 X₃ can be any amino acid. In someembodiments, the intracellular activating domain of an engineeredsignaling polypeptide includes 3 ITAM motifs.

A suitable intracellular activating domain can be an ITAMmotif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable intracellular activatingdomain can be an ITAM motif-containing domain from any ITAMmotif-containing protein. Thus, a suitable intracellular activatingdomain need not contain the entire sequence of the entire protein fromwhich it is derived. Examples of suitable ITAM motif-containingpolypeptides include, but are not limited to: CD3Z (CD3 zeta); CD3D (CD3delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (antigen receptorcomplex-associated protein alpha chain); CD79B (antigen receptorcomplex-associated protein beta chain)DAP12; and FCERlG (Fc epsilonreceptor I gamma chain).

In some embodiments, the intracellular activating domain is derived fromT cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cellreceptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).For example, a suitable intracellular activating domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids in the following sequences or to a contiguous stretchof from about 100 amino acids to about 110 amino acids (aa), from about110 aa to about 115 aa, from about 115 aa to about 120 aa, from about120 aa to about 130 aa, from about 130 aa to about 140 aa, from about140 aa to about 150 aa, or from about 150 aa to about 160 aa, of eitherof the following amino acid sequences (2 isoforms):MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID N026) orMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR(SEQ ID NO:27), where the ITAM motifs are set out with brackets.

Likewise, a suitable intracellular activating domain polypeptide caninclude an ITAM motif-containing a portion of the full length CD3 zetaamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequences or to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, or from about 150 aa toabout 160 aa, of either of the following amino acid sequences:RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO:28);RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR (SEQ ID NO:29);NQL[YNELNLGRREEYDVL]DKR SEQ ID NO:30); EGL[YNELQKDKMAEAYSEI]GMK (SEQ IDNO:31); or DGL[YQGLSTATKDTYDAL]HMQ (SEQ ID NO:32), where the ITAM motifsare set out in brackets.

In some embodiments, the intracellular activating domain is derived fromT cell surface glycoprotein CD3 delta chain (also known as CD3D;CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen,delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3delta chain; T cell surface glycoprotein CD3 delta chain; etc.). Thus, asuitable intracellular activating domain can include a domain with atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a stretch of at least 10, 15, 20, or all aminoacids in the following sequences or to a contiguous stretch of fromabout 100 amino acids to about 110 amino acids (aa), from about 110 aato about 115 aa, from about 115 aa to about 120 aa, from about 120 aa toabout 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 160 aa, of either of thefollowing amino acid sequences:MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO:33) orMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSrrWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ IDNO:34), where the ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 deltaamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:DQV[YQPLRDRDDAQYSHL]GGN (SEQ ID NO:35), where the ITAM motifs are setout in brackets.

In some embodiments, the intracellular activating domain is derived fromT cell surface glycoprotein CD3 epsilon chain (also known as CD3e, Tcell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoproteinCD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). Thus, asuitable intracellular activating domain can include a domain with atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a stretch of at least 10, 15, 20, or all aminoacids in the following sequences or to a contiguous stretch of fromabout 100 amino acids to about 110 amino acids (aa), from about 110 aato about 115 aa, from about 115 aa to about 120 aa, from about 120 aa toabout 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 160 aa, of the followingamino acid sequence:MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD[YEPIRKGQRDLYSGL]NQRRI (SEQ ID NO:36), where the ITAM motifs are set out inbrackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 epsilonamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96% 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:NPD[YEPIRKGQRDLYSGL]NQR (SEQ ID NO:37), where the ITAM motifs are setout in brackets.

In some embodiments, the intracellular activating domain is derived fromT cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cellreceptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3complex), etc.). Thus, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequences or to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, or from about 150 aa toabout 160 aa, of the following amino acid sequence:MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL[YQPLKDREDDQYSHL]QGNQLRRN (SEQ ID NO:38),where the ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 gammaamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 9790, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:DQL[YQPLKDREDDQYSHL]QGN (SEQ ID NO:39), where the ITAM motifs are setout in brackets.

In some embodiments, the intracellular activating domain is derived fromCD79A (also known as B-cell antigen receptor complex-associated proteinalpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1membrane glycoprotein; Ig-alpha; membrane-boundimmunoglobulin-associated protein; surface IgM-associated protein;etc.). Thus, a suitable intracellular activating domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids in the following sequences or to a contiguous stretchof from about 100 amino acids to about 110 amino acids (aa), from about110 aa to about 115 aa from about 115 aa to about 120 aa from about 120aa to about 130 aa from about 130 aa to about 140 aa from about 140 aato about 150 aa or from about 150 aa to about 160 aa of either of thefollowing amino acid sequences:MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAUFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLUQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:40) orMPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAWPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ IDNO:41), where the ITAM motifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD79A aminoacid sequence. Thus, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequence:ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO:42), where the ITAM motifs are setout in brackets.

In some embodiments, the intracellular activating domain is derived fromDAP12 (also known as TYROBP; TYRO protein tyrosine kinase bindingprotein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associatedprotein; TYRO protein tyrosine kinase-binding protein; killer activatingreceptor associated protein; killer-activating receptor-associatedprotein; etc.). For example, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96% 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequences or to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, or from about 150 aa toabout 160 aa, of either of the following amino acid sequences (4isoforms):MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO:43),MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO:44),MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID N045), orMGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO:46), where the ITAMmotifs are set out in brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length DAP12 aminoacid sequence. Thus, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequence:ESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO:47), where the ITAM motifs are setout in brackets.

In some embodiments, the intracellular activating domain is derived fromFCERlG (also known as FCRG; Fc epsilon receptor I gamma chain; Fcreceptor gamma-chain; fc-epsilon RJ-gamma; fcRgamma; fceRI gamma; highaffinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin Ereceptor, high affinity, gamma chain; etc.). For example, a suitableintracellular activating domain can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all amino acids in thefollowing sequences or to a contiguous stretch of from about 50 aminoacids to about 60 amino acids (aa), from about 60 aa to about 70 aa,from about 70 aa to about 80 aa, or from about 80 aa to about 88 aa, ofthe following amino acid sequence:MIPAWLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV[YTGLSTRNQETYETL]KHEKPPQ (SEQ ID NO:48), where the ITAM motifs are set outin brackets.

Likewise, a suitable intracellular activating domain polypeptide cancomprise an ITAM motif-containing portion of the full length FCER1Gamino acid sequence. Thus, a suitable intracellular activating domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all amino acids in the following sequence:DGV[YTGLSTRNQETYETL]KHE (SEQ ID NO:49), where the ITAM motifs are setout in brackets.

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure include a DAP10/CD28type signaling chain. An example of a DAP10 signaling chain is the aminoacid SEQ ID NO:50. In some embodiments, a suitable intracellularactivating domain includes a domain with at least 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to astretch of at least 10, 15, 20, or all amino acids in SEQ ID NO:50.

An example of a CD28 signaling chain is the amino acid sequence is SEQID NO:51. In some embodiments, a suitable intracellular domain includesa domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids of SEQ ID NO:51.

Intracellular activating domains suitable for use in an engineeredsignaling polypeptide of the present disclosure include a ZAP70polypeptide. For example, a suitable intracellular activating domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all amino acids in the following sequences or to acontiguous stretch of from about 300 amino acids to about 400 aminoacids, from about 400 amino acids to about 500 amino acids, or fromabout 500 amino acids to 619 amino acids, of SEQ ID NO:52.

Modulatory Domains

Modulatory domains can change the effect of the intracellular activatingdomain in the engineered signaling poly peptide, including enhancing ordampening the downstream effects of the activating domain or changingthe nature of the response. Modulatory domains suitable for use in anengineered signaling polypeptide of the present disclosure includeco-stimulatory domains. A modulatory domain suitable for inclusion inthe engineered signaling polypeptide can have a length of from about 30amino acids to about 70 amino acids (aa), e.g., a modulatory domain canhave a length of from about 30 aa to about 35 aa, from about 35 aa toabout 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa,from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. Inother cases, modulatory domain can have a length of from about 70 aa toabout 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

Co-stimulatory domains typically enhance and/or change the nature of theresponse to an activation domain. Co-stimulatory domains suitable foruse in an engineered signaling polypeptide of the present disclosure aregenerally polypeptides derived from receptors. In some embodiments,co-stimulatory domains homodimerize. A subject co-stimulatory domain canbe an intracellular portion of a transmembrane protein (i.e., theco-stimulatory domain can be derived from a transmembrane protein).Non-limiting examples of suitable co-stimulatory polypeptides include,but are not limited to, 4-lBB (CD137), CD27, CD28, CD28 deleted for Lckbinding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. Forexample, a co-stimulatory domain of an aspect of the invention can haveat least 80%, 90%, or 95% sequence identity to the co-stimulatory domainof 4-lBB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICΔ), ICOS,OX40, BTLA, CD27, CD30, GITR, or HVEM. For example, a co-stimulatorydomain of an aspect of the invention can have at least 80%, 90%, or 95%sequence identity to the co-stimulatory domain of non-limiting examplesof suitable co-stimulatory polypeptides include, but are not limited to,4-lBB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICΔ), ICOS,OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, a co-stimulatorydomain of an aspect of the invention can have at least 80%, 90%, or 95%sequence identity to the co-stimulatory domain of 4-lBB (CD137), CD27,CD28, CD28 deleted for Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30,GITR, or HVEM.

A co-stimulatory domain suitable for inclusion in an engineeredsignaling polypeptide can have a length of from about 30 amino acids toabout 70 amino acids (aa), e.g., a co-stimulatory domain can have alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In othercases, the co-stimulatory domain can have a length of from about 70 aato about 100 aa, from about 100 aa to about 200 aa, or greater than 200aa.

In some embodiments, tire co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD137 (also known asTNFRSF9; CD137; 4-lBB; CDwl37; ILA; etc.). For example, a suitableco-stimulatory domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:53. In some of these embodiments, the co-stimulatory domain has alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD28 (also known asTp44). For example, a suitable co-stimulatory domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NO:54. In some of these embodiments,the co-stimulatory domain has a length of from about 30 aa to about 35aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, or fromabout 65 aa to about 70 aa.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD28 deleted for Lckbinding (ICΔ). For example, a suitable co-stimulatory domain can includea domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NO:55. In some of these embodiments,the co-stimulatory domain has a length of from about 30 aa to about 35aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, or fromabout 65 aa to about 70 aa.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein ICOS (also known asAILIM, CD278, and CVIDl). For example, a suitable co-stimulatory domaincan include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of atleast 10, 15, 20, or all of the amino acids in SEQ ID NO:56. In some ofthese embodiments, the co-stimulatory domain has a length of from about30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aato about 45 aa, from about 45 aa to about 50 aa, from about 50 aa toabout 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about65 aa, or from about 65 aa to about 70 aa.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein OX40 (also known asTNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGPlL). OX40 contains a p85PI3K binding motif at residues 34-57 and a TRAF binding motif atresidues 76-102, each of SEQ ID NO: 296 (of Table 1). In someembodiments, the costimulatory domain can include the p85 PI3K bindingmotif of OX40. In some embodiments, the costimulatory domain can includethe TRAF binding motif of OX40. Lysines corresponding to amino acids 17and 41 of SEQ ID NO: 296 are potentially negative regulatory sites thatfunction as parts of ubiquitin targeting motifs. In some embodiments,one or both of these Lysines in the co stimulatory domain of OX40 aremutated Arginines or another amino acid. In some embodiments, a suitableco-stimulatory domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:57. In some of these embodiments, the co-stimulatory domain has alength of from about 20 aa to about 25 aa, about 25 aa to about 30 aa,30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aato about 45 aa, or from about 45 aa to about 50 aa. In illustrativeembodiments, the co-stimulatory domain has a length of from about 20 aato about 50 aa, for example 20 aa to 45 aa, or 20 aa to 42 aa.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD27 (also known as S152, T 14, TNFRSF7, and Tp55). For example, a suitable co-stimulatorydomain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch ofat least 10, 15, 20, or all of the amino acids in SEQ ID NO:58. In someof these embodiments, the co-stimulatory domain has a length of fromabout 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about40 aa to about 45 aa, or from about 45 aa to about 50 aa.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein BTLA (also known asBTLAl and CD272). For example, a suitable co-stimulatory domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% 99% or 100% sequence identity to a stretch of at least 10,15, 20, or all of the amino acids in SEQ ID NO:59.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD30 (also known asTNFRSF8, DlS166E, and Ki-1). For example, a suitable co-stimulatorydomain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96% 97%, 98%, 99% or 100% sequence identity to a stretch offrom about 100 amino acids to about 110 amino acids (aa), from about 110aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aato about 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, from about 150 aa to about 160 aa, or from about 160 aa toabout 185 aa of SEQ ID NO:60.

In some embodiments, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein GITR (also known asTNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitableco-stimulatory domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:61. In some of these embodiments, the co-stimulatory domain has alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

In some embodiments, the co-stimulatory domain derived from anintracellular portion of the transmembrane protein HVEM (also known asTNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). Forexample, a suitable co-stimulatory domain can include a domain with atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to a stretch of at least 10, 15, 20, or all of theamino acids in SEQ ID NO:62. In some of these embodiments, theco-stimulatory domain of both the first and the second polypeptide has alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa.

Linker

In some embodiments, the engineered signaling polypeptide includes alinker between any two adjacent domains. For example, a linker can bebetween the transmembrane domain and the first co-stimulatory domain. Asanother example, the ASTR can be an antibody and a linker can be betweenthe heavy chain and the light chain. As another example, a linker can bebetween the ASTR and the transmembrane domain and a co-stimulatorydomain. As another example, a linker can be between the co-stimulatorydomain and the intracellular activating domain of the secondpolypeptide. As another example, the linker can be between the ASTR andthe intracellular signaling domain.

The linker peptide may have any of a variety of amino acid sequences.Proteins can be joined by a spacer peptide, generally of a flexiblenature, although other chemical linkages are not excluded. A linker canbe a peptide of between about 1 and about 100 amino acids in length, orbetween about 1 and about 25 amino acids in length. These linkers can beproduced by using synthetic, linker-encoding oligonucleotides to couplethe proteins. Peptide linkers with a degree of flexibility can be used.The linking peptides may have virtually any amino acid sequence, bearingin mind that suitable linkers will have a sequence that results in agenerally flexible peptide. The use of small amino acids, such asglycine and alanine, are of use in creating a flexible peptide. Thecreation of such sequences is routine to those of skill in the art.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n),GGGS_(n), and GGGGS_(n) where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers are ofinterest since both of these amino acids are relatively unstructured,and therefore may serve as a neutral tether between components. Glycinepolymers are of particular interest since glycine accesses significantlymore phi-psi space than even alanine, and is much less restricted thanresidues with longer side chains (see Scheraga, Rev Computational Chem.11173-142 (1992)). Exemplary flexible linkers include, but are notlimited GGGGSGGGGSGGGGS (SEQ ID NO:63), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:64), GGGGSGGGSGGGGS (SEQ ID NO:65), GGSG (SEQ ID NO:66),GGSGG (SEQ ID NO:67), GSGSG (SEQ ID NO:68), GSGGG (SEQ ID NO:69), GGGSG(SEQ ID NO:70), GSSSG (SEQ ID NO:71), and the like. The ordinarilyskilled artisan will recognize that design of a peptide conjugated toany elements described above can include linkers that are all orpartially flexible, such that the linker can include a flexible linkeras well as one or more portions that confer less flexible structure.

Combinations

In some embodiments, a polynucleotide provided by the replicationincompetent recombinant retroviral particles has one or moretranscriptional units that encode certain combinations of the one ormore engineered signaling polypeptides. In some methods and compositionsprovided herein, genetically modified T cells include the combinationsof the one or more engineered signaling polypeptides after transductionof T cells by the replication incompetent recombinant retroviralparticles. It will be understood that the reference of a firstpolypeptide, a second polypeptide, a third polypeptide, etc. is forconvenience and elements on a “first polypeptide” and those on a “secondpolypeptide” means that the elements are on different polypeptides thatare referenced as first or second for reference and convention only,typically in further elements or steps to that specific polypeptide.

In some embodiments, the first engineered signaling polypeptide includesan extracellular antigen binding domain, which is capable of binding anantigen, and an intracellular signaling domain. In other embodiments,the first engineered signaling polypeptide also includes a T cellsurvival motif and/or a transmembrane domain. In some embodiments, thefirst engineered signaling polypeptide does not include a co-stimulatorydomain, while in other embodiments, the first engineered signalingpolypeptide does include a co-stimulatory domain.

In some embodiments, a second engineered signaling polypeptide includesa lymphoproliferative gene product and optionally an extracellularantigen binding domain. In some embodiments, the second engineeredsignaling polypeptide also includes one or more of the following: a Tcell survival motif, an intracellular signaling domain, and one or moreco-stimulatory domains. In other embodiments, when two engineeredsignaling polypeptides are used, at least one is a CAR.

In one embodiment, the one or more engineered signaling polypeptides areexpressed under a T cell specific promoter or a general promoter underthe same transcript wherein in the transcript, nucleic acids encodingthe engineered signaling polypeptides are separated by nucleic acidsthat encode one or more internal ribosomal entry sites (IREs) or one ormore protease cleavage peptides.

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes a first extracellular antigen binding domain, whichis capable of binding to a first antigen, and a first intracellularsignaling domain but not a co-stimulatory domain, and the secondpolypeptide includes a second extracellular antigen binding domain,which is capable of binding VEGF, and a second intracellular signalingdomain, such as for example, the signaling domain of a co-stimulatorymolecule. In a certain embodiment, the first antigen is PSCA, PSMA, orBCMA. In a certain embodiment, the first extracellular antigen bindingdomain comprises an antibody or fragment thereof (e.g., scFv), e.g., anantibody or fragment thereof specific to PSCA, PSMA, or BCMA. In acertain embodiment, the second extracellular antigen binding domain thatbinds VEGF is a receptor for VEGF, i.e., VEGFR. In certain embodiments,the VEGFR is VEGFR1, VEGFR2, or VEGFR3. In a certain embodiment, theVEGFR is VEGFR2.

In certain embodiments, tire polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes an extracellular tumor antigen binding domain and aCD3ζ signaling domain, and the second engineered signaling polypeptideincludes an antigen-binding domain, wherein tire antigen is anangiogenic or vasculogenic factor, and one or more co-stimulatorymolecule signaling domains. The angiogenic factor can be, e.g., VEGF.The one or more co-stimulatory molecule signaling motifs can comprise,e.g., co-stimulatory signaling domains from each of CD27, CD28, OX40,ICOS, and 4-1BB.

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes an extracellular tumor antigen-binding domain and aCD3ζ signaling domain, the second polypeptide comprises anantigen-binding domain, which is capable of binding to VEGF, andco-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS,and 4-1BB. In a further embodiment, the first signaling polypeptide orsecond signaling polypeptide also has a T cell survival motif. In someembodiments, the T cell survival motif is, or is derived from, anintracellular signaling domain of IL-7 receptor (IL-7R), anintracellular signaling domain of IL-12 receptor, an intracellularsignaling domain of IL-15 receptor, an intracellular signaling domain ofIL-21 receptor, or an intracellular signaling domain of transforminggrowth factor μ (TGFβ) receptor or the TGFβ decoy-receptor(TGF-β-dominant-negative receptor II (DNRII)).

In certain embodiments, the polynucleotide encodes two engineeredsignaling polypeptides wherein the first engineered signalingpolypeptide includes an extracellular tumor antigen-binding domain and aCD3ζ signaling domain, and the second engineered signaling polypeptideincludes an antigen-binding domain, which is capable of binding to VEGF,an IL-7 receptor intracellular T cell survival motif, and co-stimulatorysignaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.

In some embodiments, more titan two signaling polypeptides are encodedby the polynucleotide. In certain embodiments, only one of theengineered signaling polypeptides includes an antigen binding domainthat binds to a tumor-associated antigen or a tumor-specific antigen;each of the remainder of the engineered signaling polypeptides comprisesan antigen binding domain that binds to an antigen that is not atumor-associated antigen or a tumor-specific antigen. In otherembodiments, two or more of the engineered signaling polypeptidesinclude antigen binding domains that bind to one or moretumor-associated antigens or tumor-specific antigens, wherein at leastone of the engineered signaling polypeptides comprises an antigenbinding domain that does not bind to a tumor-associated antigen or atumor-specific antigen.

In some embodiments, the tumor-associated antigen or tumor-specificantigen is Her2, prostate stem cell antigen (PSCA), PSMA(prostate-specific membrane antigen), B cell maturation antigen (BCMA),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancerantigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membraneprotein (EMA), epithelial tumor antigen (ETA), tyrosinase,melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117,chromogranin, cytokeratin, desmin, glial fibrillary acidic protein(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen,protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1),myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specificenolase (NSE), placental alkaline phosphatase, synaptophysin,thyroglobulin, thyroid transcription factor-1, the dimeric form of thepyruvate kinase isoenzyme type M2 (tumor M2-PK), CD19, CD22, CD27, CD30,CD70, GD2 (ganglioside G2), EphA2, CSPG4, CD138, FAP (FibroblastActivation Protein), CD171, kappa, lambda, 5T4, αvβ6 integrin, integrinαvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viraloncogene), Ral-B, B7-H3, B7-H6, CAIX, CD20, CD33, CD44, CD44v6,CD44v7/8, CD123, EGFR, EGP2, EGP40, EpCAM, fetal AchR, FRα, GD3,HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lewis-Y, Muc16, NCAM,NKG2D Ligands, NY-ESO-1, FRAME, ROR1, Survivin, TAG72, TEMs, VEGFR2,EGFRvIII (epidermal growth factor variant ID), sperm protein 17 (Sp17),mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cellreceptor gamma alternate reading frame protein), Trp-p8, STEAP1(six-transmembrane epithelial antigen of the prostate 1), an abnormalras protein, or an abnormal p53 protein.

In some embodiments, the first engineered signaling polypeptide includesa first extracellular antigen binding domain that binds a first antigen,and a first intracellular signaling domain; and a second engineeredsignaling polypeptide includes a second extracellular antigen bindingdomain that binds a second antigen, or a receptor that binds the secondantigen; and a second intracellular signaling domain, wherein the secondengineered signaling polypeptide does not comprise a co-stimulatorydomain. In a certain embodiment, the first antigen-binding domain andthe second antigen-binding domain are independently an antigen-bindingportion of a receptor o r an antigen-binding portion of an antibody. Ina certain embodiment, either or both of the first antigen binding domainor the second antigen binding domain are scFv antibody fragments. Incertain embodiments, the first engineered signaling polypeptide and/orthe second engineered signaling polypeptide additionally comprises atransmembrane domain. In a certain embodiment, the first engineeredsignaling polypeptide or the second engineered signaling polypeptidecomprises a T cell survival motif, e.g., any of the T cell survivalmotifs described herein.

In another embodiment, the first engineered signaling polypeptideincludes a first extracellular antigen binding domain that binds HER2and the second engineered signaling polypeptide includes a secondextracellular antigen binding domain that binds MUC-1.

In another embodiment, the second extracellular antigen binding domainof the second engineered signaling polypeptide binds an interleukin.

In another embodiment, the second extracellular antigen binding domainof the second engineered signaling polypeptide binds a damage associatedmolecular pattern molecule (DAMP; also known as an alarm in). In otherembodiments, a DAMP is a heat shock protein, chromatin-associatedprotein high mobility group box 1 (HMGB1), S100A8 (also known as MRP8,or calgranulin A), S100A9 (also known as MRP14, or calgranulin B), serumamyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uricacid, or heparin sulfate.

In certain embodiments, said second antigen is an antigen on an antibodythat binds to an antigen presented by a tumor cell.

In some embodiments, signal transduction activation through the secondengineered signaling polypeptide is non-antigenic, but is associatedwith hypoxia. In certain embodiments, hypoxia is induced by activationof hypoxia-inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α,or HIF-3β.

In some embodiments, expression of the one or more engineered signalingpolypeptides is regulated by a control element, which is disclosed inmore detail herein.

Additional Sequences

The engineered signaling polypeptides, such as CARs, can further includeone or more additional polypeptide domains, where such domains include,but are not limited to, a signal sequence; an epitope tag; an affinitydomain; and a polypeptide whose presence or activity can be detected(detectable marker), for example by an antibody assay or because it is apolypeptide that produces a detectable signal. Non-limiting examples ofadditional domains for any of the aspects or embodiments providedherein, include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of thefollowing sequences as described below: a signal sequence, an epitopetag, an affinity domain, or a polypeptide that produces a detectablesignal.

Signal sequences that are suitable for use in a subject CAR, e.g., inthe first polypeptide of a subject CAR, include any eukaryotic signalsequence, including a naturally-occurring signal sequence, a synthetic(e.g., man-made) signal sequence, etc. In some embodiments, for example,the signal sequence can be the CD8 signal sequence MALPVTALLLPLALLLHAARP(SEQ ID NO:72).

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., YPYDVPDYA; SEQ ID NO:73); FLAG (e.g., DYKDDDDK; SEQ IDNO:74); c-myc (e.g., EQKLISEEDL; SEQ ID NO:75), and the like.

Affinity domains include peptide sequences that can interact with abinding partner, e.g., such as one immobilized on a solid support,useful for identification or purification. DNA sequences encodingmultiple consecutive single amino acids, such as histidine, when fusedto the expressed protein, may be used for one-step purification of therecombinant protein by high affinity binding to a resin column, such asnickel sepharose. Exemplary affinity domains include His5 (HHHHH; SEQ IDNO:76), HisX6 (HHHHHH; SEQ ID NO:77), c-myc (EQKLISEEDL; SEQ ID NO:75),Flag (DYKDDDDK; SEQ ID NO:74), Strep Tag (WSHPQFEK; SEQ ID NO:78),hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:73), GST, thioredoxin,cellulose binding domain, RYIRS (SEQ ID NO:79), Phe-His-His-Thr (SEQ IDNO:80), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-endRNA tag, WEAAAREACCRECCARA (SEQ ID NO:81), metal binding domains, e.g.,zinc binding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins,pan-albumin, calbindin D9K, calbindin D28K, and calretinin, inteins,biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltosebinding protein.

Suitable detectable signal-producing proteins include, e.g., fluorescentproteins; enzymes that catalyze a reaction that generates a detectablesignal as a product; and the like.

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP),destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat.Methods 2:905-909), and the like. Any of a variety of fluorescent andcolored proteins from Anthozoan species, as described in, e.g., Matz etal. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

Suitable enzymes include, but are not limited to, horse radishperoxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL),glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase,glucose oxidase (GO), and the like.

Recognition and/or Elimination Domain

Any of the replication incompetent recombinant retroviral particlesprovided herein can include nucleic acids that encode a recognition orelimination domain as part of, or separate from, nucleic acids encodingany of the engineered signaling polypeptides provided herein. Thus, anyof the engineered signaling polypeptides provided herein, can include arecognition or elimination domain. For example, any of the CARsdisclosed herein can include a recognition or elimination domain.Moreover, a recognition or elimination domain can be expressed togetherwith, or even fused with any of the lymphoproliferative elementsdisclosed herein. The recognition or elimination domains are expressedon the T cell and/or NK cell but are not expressed on the replicationincompetent recombinant retroviral particles.

In some embodiments, the recognition or elimination domain can bederived from herpes simplex virus-derived enzyme thymidine kinase(HSV-tk) or inducible caspase-9. In some embodiments, the recognition orelimination domain can include a modified endogenous cell-surfacemolecule, for example as disclosed in U.S. Pat. No. 8,802,374. Themodified endogenous cell-surface molecule can be any cell-surfacerelated receptor, ligand, glycoprotein, cell adhesion molecule, antigen,integrin, or cluster of differentiation (CD) that is modified. In someembodiments, the modified endogenous cell-surface molecule is atruncated tyrosine kinase receptor. In one aspect, the truncatedtyrosine kinase receptor is a member of the epidermal growth factorreceptor (EGFR) family (e.g., ErbB1, ErbB2, ErbB3, and ErbB4). In someembodiments, the recognition domain can be a polypeptide that isrecognized by an antibody that recognizes the extracellular domain of anEGFR member. In some embodiments, the recognition domain can be at least20 contiguous amino acids of an EGFR family member, or for example,between 20 and 50 contiguous amino acids of an EGFR family member. Forexample, SEQ ID NO:82, is an exemplary-polypeptide that is recognizedby, and under the appropriate conditions bound by an antibody thatrecognizes the extracellular domain of an EGFR member. Suchextracellular EGFR epitopes are sometimes referred to herein as eTags.In illustrative embodiments, such epitopes are recognized bycommercially available anti-EGFR monoclonal antibodies.

Epidermal growth factor receptor, also known as EGFR, ErbB1 and HER1, isa cell-surface receptor for members of the epidermal growth factorfamily of extracellular ligands. Alterations in EGFR activity have beenimplicated in certain cancers. In some embodiments, a gene encoding anEGFR polypeptide including human epidermal growth factor receptor (EGFR)is constructed by removal of nucleic acid sequences that encodepolypeptides including the membrane distal EGF-binding domain and thecytoplasmic signaling tail, but retains the extracellular membraneproximal epitope recognized by an anti-EGFR antibody. Preferably, theantibody is a known, commercially available anti-EGFR monoclonalantibody, such as cetuximab, matuzumab, necitumumab or panitumumab.

Others have shown that application of biotinylated-cetuximab toimmunomagnetic selection in combination with anti-biotin microbeadssuccessfully enriches T cells that have been lentivirally transducedwith EGFRt-containing constructs from as low as 2% of the population togreater than 90% purity without observable toxicity to the cellpreparation. Furthermore, others have shown that constitutive expressionof this inert EGFR molecule does not affect T cell phenotype or effectorfunction as directed by the coordinately expressed chimeric antigenreceptor (CAR), CD19R. In addition, others have shown that through flowcytometric analysis, EGFR was successfully utilized as an in vivotracking marker for T cell engraftment in mice. Furthermore, EGFR wasdemonstrated to have suicide gene potential through Erbitux® mediatedantibody dependent cellular cytotoxicity (ADCC) pathways. The inventorsof the present disclosure have successfully expressed eTag in PBMCsusing lentiviral vectors, and have found that expression of eTag invitro by PBMCs exposed to Cetuximab, provided an effective eliminationmechanism for PBMCs. Thus, EGFR may be used as a non-immunogenicselection tool, tracking marker, and suicide gene for transduced T cellsthat have immunotherapeutic potential. The EGFR nucleic acid may also bedetected by means well known in the art.

In some embodiments provided herein, EGFR is expressed as part of asingle polypeptide that also includes the CAR or as part of a singlepolypeptide that includes the lymphoproliferative element. In someembodiments, the amino acid sequence encoding the EGFR recognitiondomain can be separated from the amino acid sequence encoding thechimeric antigen receptor by a cleavage signal and/or a ribosomal skipsequence. The ribosomal skip and/or cleavage signal can be any ribosomalskip and/or cleavage signal known in the art. Not to be limited bytheory, the ribosomal skip sequence can be, for example T2A (alsoreferred to as 2A-1 herein) with amino acid sequenceGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:83). Not to be limited by theory, otherexamples of cleavage signals and ribosomal skip sequences include FMDV2A (F2A); equine rhinitis A virus 2A (abbreviated as E2A); porcineteschovirus-1 2A (P2A); and Thoseaasigna virus 2A (T2A). In someembodiments, the polynucleotide sequence encoding the recognition domaincan be on the same transcript as the CAR or lymphoproliferative elementbut separated from the polynucleotide sequence encoding the CAR orlymphoproliferative element by an internal ribosome entry site.

In other embodiments as exemplified empirically herein, a recognitiondomain can be expressed as part of a fusion polypeptide, fused to alymphoproliferative element. Such constructs provide the advantage,especially in combination with other “space saving” elements providedherein, of taking up less genomic space on an RNA genome compared toseparate polypeptides. In one illustrative embodiment, an eTag isexpressed as a fusion polypeptide, fused to an IL7Rα mutant, asexperimentally demonstrated herein.

Chimeric Antigen Receptor

In some aspects of the present invention, an engineered signalingpolypeptide is a chimeric antigen receptor (CAR) or a polynucleotideencoding a CAR, which, for simplicity, is referred to herein as “CAR.” ACAR of the present disclosure includes: a) at least one antigen-specifictargeting region (ASTR); b) a transmembrane domain; and c) anintracellular activating domain. In illustrative embodiments, theantigen-specific targeting region of the CAR is an scFv portion of anantibody to the target antigen. In illustrative embodiments, theintracellular activating domain is from CD3Z, CD3D, CD3E, CD3G, CD79A,CD79B, DAP12, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70, and somefurther illustrative embodiments, from CD3z. In illustrativeembodiments, the CAR further comprises a co-stimulatory domain, forexample any of the co-stimulatory domains provided above in theModulatory Domains section, and in further illustrative embodiments theco-stimulatory domain is the intracellular co-stimulatory domain of4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. Insome embodiments, the CAR includes any of the transmembrane domainslisted in the Transmembrane Domain section above.

A CAR of the present disclosure can be present in the plasma membrane ofa eukaryotic cell, e.g., a mammalian cell, where suitable mammaliancells include, but are not limited to, a cytotoxic cell, a T lymphocyte,a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of aprogenitor cell, and an NK cell, an NK-T cell, and a macrophage. Whenpresent in the plasma membrane of a eukaryotic cell, a CAR of thepresent disclosure is active in the presence of one or more targetantigens that, in certain conditions, binds the ASTR. The target antigenis the second member of the specific binding pair. The target antigen ofthe specific binding pair can be a soluble (e.g., not bound to a cell)factor; a factor present on the surface of a cell such as a target cell;a factor presented on a solid surface; a factor present in a lipidbilayer; and the like. Where the ASTR is an antibody, and the secondmember of the specific binding pair is an antigen, the antigen can be asoluble (e.g., not bound to a cell) antigen; an antigen present on thesurface of a cell such as a target cell; an antigen presented on a solidsurface; an antigen present in a lipid bilayer; and the like.

In some instances, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, increases expression of at least one nucleic acid inthe cell. For example, in some cases, a CAR of the present disclosure,when present in the plasma membrane of a eukaryotic cell, and whenactivated by the one or more target antigens, increases expression of atleast one nucleic acid in the cell by at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 75% at least about2-fold, at least about 2.5-fold, at least about 5-fold, at least about10-fold, or more than 10-fold, compared with the level of transcriptionof the nucleic acid in the absence of the one or more target antigens.

As an example, the CAR of the present disclosure can include animmunoreceptor tyros me-based activation motif (ITAM)-containingintracellular signaling polypeptide.

A CAR of the present disclosure, when present in the plasma membrane ofa eukaryotic cell, and when activated by one or more target antigens,can, in some instances, result in increased production of one or morecytokines by the cell. For example, a CAR of the present disclosure,when present in the plasma membrane of a eukaryotic cell, and whenactivated by the one or more target antigens, can increase production ofa cytokine by the cell by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 75%, at least about 2-fold, atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, ormore than 10-fold, compared with the amount of cytokine produced by thecell in the absence of the one or more target antigens. Cytokines whoseproduction can be increased include, but are not limited to interferongamma (IFN-γ), tumor necrosis factor-alpha (TNF-a), IL-2, IL-15, IL-12,IL-4, IL-5, IL-10; a chemokine; a growth factor; and the like.

In some embodiments, a CAR of the present disclosure, when present inthe plasma membrane of a eukaryotic cell, and when activated by one ormore target antigens, can result in both an increase in transcription ofa nucleic acid in the cell and an increase in production of a cytokineby tire cell.

In some instances, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by one or moretarget antigens, results in cytotoxic activity by the cell toward atarget cell that expresses on its cell surface an antigen to which theantigen-binding domain of the first polypeptide of the CAR binds. Forexample, where the eukaryotic cell is a cytotoxic cell (e.g., an NK cellor a cytotoxic T lymphocyte), a CAR of the present disclosure, whenpresent in the plasma membrane of the cell, and when activated by theone or more target antigens, increases cytotoxic activity of the celltoward a target cell that expresses on its cell surface the one or moretarget antigens. For example, where the eukaryotic cell is an NK cell ora T lymphocyte, a CAR of the present disclosure, when present in theplasma membrane of the cell, and when activated by the one or moretarget antigens, increases cytotoxic activity of the cell by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 75%, at least about 2-fold, at least about 2.5-fold, at leastabout 5-fold, at least about 10-fold, or more than 10-fold, compared tothe cytotoxic activity of tire cell in the absence of the one or moretarget antigens.

In some embodiments, a CAR of the present disclosure, when present inthe plasma membrane of a eukaryotic cell, and when activated by one ormore target antigens, can result in other CAR activation related eventssuch as proliferation and expansion (either due to increased cellulardivision or anti-apoptotic responses).

In some embodiments, a CAR of the present disclosure, when present inthe plasma membrane of a eukaryotic cell, and when activated by one ormore target antigens, can result in other CAR activation related eventssuch as intracellular signaling modulation, cellular differentiation, orcell death.

In some embodiments, CARs of the present disclosure are microenvironmentrestricted. This property is typically the result of themicroenvironment restricted nature of the ASTR domain of the CAR. Thus,CARs of the present disclosure can have a lower binding affinity or, inillustrative embodiments, can have a higher binding affinity to one ormore target antigens under a condition(s) in a microenvironment thanunder a condition in a normal physiological environment.

In certain illustrative embodiments, CARs provided herein comprise aco-stimulators' domain in addition to an intracellular activatingdomain, wherein the co-stimulatory domain is any of the intracellularsignaling domains provided herein for lymphoproliferative elements(LEs), such as, for example, intracellular domains of CLEs. In certainillustrative embodiments, the co-stimulatory domains of CARs herein arefirst intracellular domains (P3 domains) identified herein for CLEs orP4 domains that are shown as effective intracellular signaling domainsof CLEs herein in the absence of a P3 domain. Furthermore, in certainillustrative embodiments, co-stimulatory domains of CARs can compriseboth a P3 and a P4 intracellular signaling domain identified herein forCLEs. Certain illustrative subembodiments include especially effectiveP3 and P4 partner intracellular signaling domains as identified hereinfor CLEs. In illustrative embodiments, the co-stimulatory domain isother than an ITAM-containing intracellular domain of a CAR either aspart of the co-stimulatory domain, or in further illustrativeembodiments as the only co-stimulatory domain.

In these embodiments that include a CAR with a co-stimulatory domainidentified herein as an effective intracellular domain of an LE, theco-stimulatory domain of a CAR can be any intracellular signaling domainin Table 1 provided herein. Active fragments of any of the intracellulardomains in Table 1 can be a co-stimulatory domain of a CAR. Inillustrative embodiments, the ASTR of the CAR comprises an scFV. Inillustrative embodiments, in addition to the c-stimulatory intracellulardomain of a CLE, these CARs comprise an intracellular activating domainthat in illustrative embodiments is a CD3Z, CD3D, CD3E, CD3G, CD79A,CD79B, DAP12, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 intracellularactivating domain, or in further illustrative embodiments is a CD3zintracellular activating domain.

In these illustrative embodiments, the co-stimulatory domain of a CARcan comprise an intracellular domain or a functional signaling fragmentthereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA,CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP,IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL7RA,IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2,IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R,IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR, orPRLR. In some embodiments, the co-stimulatory domain of a CAR caninclude an intracellular domain or a functional signaling fragmentthereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA,CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP,IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL9R,IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD,IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP1, MPL,MyD88, OSMR, or PRLR. In some embodiments, the co-stimulatory domain ofa CAR can include an intracellular domain or a functional fragmentthereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R,EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R,IL9R, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R,IL23R, IL27RA, IL31RA, LEPR, MPL, MyD88, or OSMR. In some embodiments,the co-stimulatory domain of a CAR can include an intracellular domainor a fragment thereof that includes a signaling domain from CSF2RB,CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA,IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL13RA2, IL17RD, IL31RA, LEPR,MPL, MyD88, or OSMR. In some embodiments, the co-stimulatory domain of aCAR can include an intracellular domain or a functional signalingfragment thereof that includes a signaling domain from CSF2RB, CSF3R,IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RC, IL17RE,IL18R1, IL27RA, IL31RA, MPL, MyD88, OSMR, or PRLR. In some embodiments,the co-stimulatory domain of a CAR can include an intracellular domainor a functional signaling fragment thereof that includes a signalingdomain from CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE,IL31RA, MPL, or MyD88.

In some embodiments, the co-stimulatory domain of a CAR can include anintracellular domain or a fragment thereof that includes a signalingdomain from CSF3R, IL6ST, IL27RA, MPL, and MyD88. In certainillustrative subembodiments, the intracellular activating domain of theCAR is derived from CD3z.

Recombinant T Cell Receptors (TCRs)

T Cell Receptors (TCRs) recognize specific protein fragments derivedfrom intracellular and well as extracellular proteins. When proteins arebroken into peptide fragments, they are presented on the cell surfacewith another protein called major histocompatibility complex, or MHC,which is called the HLA (human leukocyte antigen) complex in humans.Three different T cell antigen receptors combinations in vertebrates areαβ TCR, γδTCR and pre-TCR. Such combinations are formed by dimerizationbetween members of dimerizing subtypes, such as an α TCR subunit and a βTCR subunit, a γ TCR subunit and a δ TCR subunit, and for pre-TCRs, apTα subunit and a β TCR subunit. A set of TCR subunits dimerize andrecognize a target peptide fragment presented in the context of an MHC.The pre-TCR is expressed only-on the surface of immature αβ T cellswhile the αβ TCR is expressed on the surface of mature αβ T cells and NKT cells, and γδTCR is expressed on the surface of γδT cells. αβTCRs onthe surface of a T cell recognize the peptide presented by MHCI or MHCIIand the αβ TCR on the surface of NK T cells recognize lipid antigenspresented by CD1. γδTCRs can recognize MHC and MHC-like molecules, andcan also recognize non-MHC molecules such as viral glycoproteins. Uponligand recognition, αβTCRs and γδTCRs transmit activation signalsthrough the CD3zeta chain that stimulate T cell proliferation andcytokine secretion.

TCR molecules belong to the immunoglobulin superfamily with itsantigen-specific presence in the V region, where CDR3 has morevariability than CDR1 and CDR2, directly determining the antigen bindingspecificity of the TCR. When the MHC-antigen peptide complex isrecognized by a TCR, the CDR1 and CDR2 recognize and bind the sidewallof the MHC molecule antigen binding channel, and the CDR3 binds directlyto the antigenic peptide. Recombinant TCRs may thus be engineered thatrecognize a tumor-specific protein fragment presented on MHC.

Recombinant TCR's such as those derived from human TCRα and TCRβ pairsthat recognize specific peptides with common HLAs can thus be generatedwith specificity to a tumor specific protein (Schmitt, T M et al.,2009). The target of recombinant TCRs may be peptides derived from anyof the antigen targets for CAR ASTRs provided herein, but are morecommonly derived from intracellular tumor specific proteins such asoncofetal antigens, or mutated variants of normal intracellular proteinsor other cancer specific neoepitopes. Libraries of TCR subunits may bescreened for their selectivity to a target antigen. Screens of naturaland/or recombinant TCR subunits can identify sets of TCR subunits withhigh avidities and/or reactivities towards a target antigen. Members ofsuch sets of TCR subunits can be selected and cloned to produce one ormore poly-nucleotide encoding the TCR subunit.

Polynucleotides encoding such a set of TCR subunits can be included in areplication incompetent recombinant retroviral particle to geneticallymodify a lymphocyte, or in illustrative embodiments, a T cell or an NKcell, such that the lymphocyte expresses the recombinant TCR.Accordingly, in any aspect or embodiment provided herein that includesan engineered signaling polypeptide, such as embodiments that includeone more CARs and/or lymphoproliferative elements, the engineeredsignaling polypeptide(s) can include or can be one or more sets ofrecombinant γδTCR chains, or in illustrative embodiments αβTCR chains.TCR chains that form a set may be co-expressed using a number ofdifferent techniques to co-express the two TCR chains as is disclosedherein for expressing two or more other engineered signalingpoly-peptides such as CARs and lymphoproliferative elements. Forexample, protease cleavage epitopes such as 2A protease, internalribosomal entry sites (IRES), and separate promoters may be used.

Several strategies have been employed to reduce the likelihood of mixedTCR dimer formation. In general, this involves modification of theconstant (C) domains of the TCRα and TCRβ chains to promote thepreferential pairing of the introduced TCR chains with each other, whilerendering them less likely to successfully pair with endogenous TCRchains. One approach that has shown some promise in vitro involvesreplacement of the C domain of human TCRα and TCRβ chains with theirmouse counterparts. Another approach involves mutation of the human TCRαcommon domain and TCRβ chain common regions to promote self-pairing, orthe expression of an endogenous TCR alpha and TCR beta miRNA within theviral gene construct. Accordingly, in some embodiments provided hereinthat include one or more sets of TCR chains as engineered signalingpolypeptides, each member of the set of TCR chains, in illustrativeembodiments αβTCR chains, comprises a modified constant domain thatpromotes preferential pairing with each other. In some subembodiments,each member of a set of TCR chains, in illustrative embodiments αβTCRchains, comprises a mouse constant domain from the same TCR chain type,or a constant domain from the same TCR chain subtype with enoughsequences derived from a mouse constant domain from the same TCR chainsubtype, such that dimerization of the set of TCR chains to each otheris preferred over, or occurs to the exclusion of, dimerization withhuman TCR chains. In other subembodiments, each member of a set of TCRchains, in illustrative embodiments αβTCR chains, comprisescorresponding mutations in its constant domain, such that dimerizationof the set of TCR chains to each other is preferred over, or occurs tothe exclusion of, dimerization with TCR chains that have human constantdomains. Such preferred or exclusive dimerization in illustrativeembodiments, is under physiological conditions.

Lymphoproliferative Elements

Peripheral T lymphocyte numbers are maintained at remarkably stablelevels throughout adulthood, despite the continuing addition of cells,due to emigration from the thymus and proliferation in response toantigen encounter, and loss of cells owing to the removal ofantigen-specific effectors after antigen clearance (Marrak, P. et al.2000. Nat Immunol 1:107-111; Freitas, A. A. et al. 2000. Annu RevImmunol 18:83-111). The size of the peripheral T cell compartment isregulated by multiple factors that influence both proliferation andsurvival. However, in a lymphopenic environment T lymphocytes divideindependently of cognate antigen, due to “acute homeostaticproliferation” mechanisms that maintain the size of the peripheral Tcell compartment. Conditions for lymphopenia have been established insubjects or patients during adoptive cell therapy by proliferating Tcells in vitro and introducing them into lymphodepleted subjects,resulting in enhanced engraftment and antitumor function of transferredT cells. However, lymphodepletion of a subject is not desirable becauseit can cause serious side effects, including immune dysfunction anddeath.

Studies have shown that lymphodepletion removes endogenous lymphocytesfunctioning as cellular sinks for homeostatic cytokines, thereby fleeingcytokines to induce survival and proliferation of adoptively transferredcells. Some cytokines, such as for example, IL-7 and IL-15, are known tomediate antigen-independent proliferation of T cells and are thuscapable of eliciting homeostatic proliferation in non-lymphopenicenvironments. However, these cytokines and their receptors haveintrinsic control mechanisms that prevent lymphoproliferative disordersat homeostasis.

Many of the embodiments provided herein include a lymphoproliferativeelement or a nucleic acid encoding the same, typically as part of anengineered signaling polypeptide. Accordingly, in some aspects of thepresent invention, an engineered signaling polypeptide is alymphoproliferative element (LE) such as a chimeric lymphoproliferativeelement (CLE). Typically, the LE comprises an extracellular domain, atransmembrane domain, and at least one intracellular signaling domainthat drives proliferation, and in illustrative embodiments a secondintracellular signaling domain.

In some embodiments, tire lymphoproliferative element can include afirst and/or second intracellular signaling domain. In some embodiments,the first and/or second intracellular signaling domain can include CD2,CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, mutated Delta Lck CD28, CD28,CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCER1G, FCGR2C,FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1,IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R,IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1,IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1,IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR,LIFR, LMP1, MPL, MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14,or TNFRSF18, or functional mutants and/or fragments thereof. Inillustrative embodiments, the first intracellular signaling domain caninclude MyD88, or a functional mutant and/or fragment thereof. Infurther illustrative embodiments, the first intracellular signalingdomain can include MyD88, or a functional mutant and/or fragmentthereof, and the second intracellular signaling domain can include ICOS,TNFRSF4, or TNSFR18, or functional mutants and/or fragments thereof. Insome embodiments, the first intracellular domain is MyD88 and the secondintracellular domain is an ITAM-containing intracellular domain, forexample, an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A,CD79B, DAP12, FCERlG, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70. In someembodiments, the second intracellular signaling domain can includeTNFRSF18, or a functional mutant and/or fragment thereof.

In some embodiments, the lymphoproliferative element can include afusion of an extracellular domain and a transmembrane domain. In someembodiments, the fusion of an extracellular domain and a transmembranedomain can include eTAG IL7RA Ins PPCL (interleukin 7 receptor), MycLMP1, LMP1, eTAG CRLF2, eTAG CSF2RB, eTAG CSF3R, eTAG EPOR, eTAG GHR,eTAG truncated after Fn F523C IL27RA, or eTAG truncated after Fn S505NMPL, or functional mutants and/or fragments thereof. In someembodiments, the lymphoproliferative element can include anextracellular domain. In some embodiments, the extracellular domain caninclude eTag with 0, 1, 2, 3, or 4 additional alanines at the carboxyterminus. In some embodiments, the extracellular domain can include Mycwith 0, 1, 2, 3, or 4 additional alanines at the carboxy terminus, orfunctional mutants and/or fragments thereof.

In some embodiments, the lymphoproliferative element can include atransmembrane domain. In some embodiments, the transmembrane domain caninclude CD2, CD3D, CD3E, CD3G, CD3Z CD247, CD4, CD8A, CD8B, CD27, CD28,CD40, CD79A, CD79B, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, FCER1G, FCGR2C,FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1,IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R,IL6ST, IL7RA, IL7RA Ins PPCL, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD,IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA,IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9,TNFRSF14, or TNFRSF18, or functional mutants and/or fragments thereof.

CLEs for use in any aspect or embodiment herein can include any CLEdisclosed in WO2019/055946 (incorporated by reference herein, in itsentirety), the vast majority of which were designed to be and arebelieved to be constitutively active. As illustrated therein, wherethere is a first and a second intracellular signaling domain of a CLE,the first intracellular signaling domain is positioned between themembrane associating motif and the second intracellular domain.

In another embodiment, the LE provides, is capable of providing and/orpossesses the property of (or a cell genetically modified and/ortransduced with the LE is capable of providing, is adapted for,possesses the property of, and/or is modified for) driving T cellexpansion in vivo. Methods for performing such an in vivo test areprovided in Example 6. For example, as illustrated in Example 6, the invivo test can utilize a mouse model and measure T cell expansion at 15to 25 days in vivo, or at 19 to 21 days in vivo, or at approximately 21days in vivo, after T cells are contacted with lentiviral vectorsencoding the LEs, are introduced into the mice.

In some embodiments, the lymphoproliferative element can include any ofthe sequences listed in Table 1 (SEQ ID NOs: 84-302). Table 1 shows theparts, names (including gene names), and amino acid sequences fordomains that were tested in CLEs. Typically, a CLE includes anextracellular domain (denoted P1), a transmembrane domain (denoted P2),a first intracellular domain (denoted P3), and a second intracellulardomain (denoted P4). Typically, the lymphoproliferative element includesa first intracellular domain. In illustrative embodiments, the firstintracellular domain can include any of the parts listed as S036 toS0216 or in Table 1, or functional mutants and/or fragments thereof. Insome embodiments, the lymphoproliferative element can include a secondintracellular domain. In illustrative embodiments, the secondintracellular domain can include any of the parts listed as S036 toS0216 or in Table 1, or functional mutants and/or fragments thereof. Insome embodiments, the lymphoproliferative element can include anextracellular domain. In illustrative embodiments, the extracellulardomain can include any of the sequences of parts listed as M001 to M049or E006 to E015 in Table 1, or functional mutants and/or fragmentsthereof. In some embodiments, the lymphoproliferative element caninclude a transmembrane domain. In illustrative embodiments, thetransmembrane domain can include any of the parts listed as M001 to M049or T001 to T082 in Table 1, or functional mutants and/or fragmentsthereof. In some embodiments, the lymphoproliferative element can be offusion of an extracellular/transmembrane domain (M001 to M049 in Table1), a first intracellular domain (S036 to S0216 in Table 1), and asecond intracellular domain (S036 to S216 in Table 1). In someembodiments, the lymphoproliferative element can be a fusion of anextracellular domain (E006 to E015 in Table 1), a transmembrane domain(T001 to T082 in Table 1), a first intracellular domain (S036 to S0216in Table 1), and a second intracellular domain (S036 to S0216 in Table1). For example, the lymphoproliferative element can be a fusion ofE006, T001, S036, and S216, also written as E006-T001-S036-S216). Inillustrative embodiments, the lymphoproliferative element can be thefusion E010-T072-S192-S212, E007-T054-S197-S212, E006-T006-S194-S211,E009-T073-S062-S053, E008-T001-S121-S212, E006-T044-S186-S053, orE006-T016-SI 86-S050.

In illustrative embodiments, the intracellular domain of an LE, or thefirst intracellular domain in an LE that has two or more intracellulardomains, is other than a functional intracellular activating domain froman ITAM-containing intracellular domain, for example, an intracellulardomain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCERlG, FCGR2A,FCGR2C, DAP10/CD28, or ZAP70, and in a further illustrativesubembodiment, CD3z. In illustrative embodiments, a second intracellulardomain of an LE is other than a co-stimulatory domain of 4-1BB (CD137),CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In illustrativeembodiments, the extracellular domain of an LE docs not comprise asingle-chain variable fragment (scFv). In further illustrativeembodiments, the extracellular domain of an LE that upon binding to abinding partner activates an LE, does not comprise a single-chainvariable fragment (scFv).

A CLE does not comprise both an ASTR and an activation domain from CD3Z,CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCERlG, FCGR2A, FCGR2C,DAP10/CD28, or ZAP70. Not to be limited by theory, the extracellulardomain and transmembrane domain are believed to play support roles inLEs, assuring that the intracellular signaling domain(s) is in aneffective conformation/orientation/localization for drivingproliferation. Thus, the ability of an LE to drive proliferation isbelieved to be provided by the intracellular domain(s) of the LE, andthe extracellular and transmembrane domains are believed to playsecondary roles relative to the intracellular domain(s). Alymphoproliferative element includes an intracellular domain that is asignaling polypeptide that is capable of driving proliferation of Tcells or NK cells that is associated with a membrane through amembrane-associating motif (e.g. a transmembrane domain) and is orientedin, or capable of being oriented into, an active conformation. The ASTRof an LE in illustrative embodiments, does not include an scFv.Strategies are provided herein for associating an intracellular domainwith a membrane, such as by inclusion of a transmembrane domain, a GPIanchor, a myristoylation region, a palmitoylation region, and/or aprenylation region. In some embodiments, a lymphoproliferative elementdoes not include an extracellular domain.

The extracellular domains, transmembrane domains, and intracellulardomains of LEs can vary in their respective amino acid lengths. Forexample, for embodiments that include a replication incompetentretroviral particle, there are limits to the length of a polynucleotidethat can be packaged into a retroviral particle so LEs with shorteramino acid sequences can be advantageous in certain illustrativeembodiments. In some embodiments, the overall length of the LE can bebetween 3 and 4000 amino acids, for example between 10 and 3000, 10 and2000, 50 and 2000, 250 and 2000 amino acids, and, in illustrativeembodiments between 50 and 1000, 100 and 1000 or 250 and 1000 aminoacids. The extracellular domain, when present to form an extracellularand transmembrane domain, can be between 1 and 1000 amino acids, and istypically between 4 and 400, between 4 and 200, between 4 and 100,between 4 and 50, between 4 and 25, or between 4 and 20 amino acids. Inone embodiment, the extracellular region is GGGS for an extracellularand transmembrane domain of this aspect of the invention. Thetransmembrane domains, or transmembrane regions of extracellular andtransmembrane domains, can be between 10 and 250 amino acids, and aremore typically at least 15 amino acids in length, and can be, forexample, between 15 and 100, 15 and 75, 15 and 50, 15 and 40, or 15 and30 amino acids in length. The intracellular signaling domains can be,for example, between 10 and 1000, 10 and 750, 10 and 500, 10 and 250, or10 and 100 amino acids. In illustrative embodiments, the intracellularsignaling domain can be at least 30, or between 30 and 500, 30 and 250,30 and 150, 30 and 100, 50 and 500, 50 and 250, 50 and 150, or 50 and100 amino acids. In some embodiments, an intracellular signaling domainfor a particular gene is at least 90%, 95%, 98%, 99% or 100% identicalto at least 10, 25, 30, 40, or 50 amino acids from a sequence of thatintracellular signaling domain, such as a sequence provided herein forthat intracellular domain, up to the size of tire entire intracellulardomain sequence, and can include for example, up to an additional 1, 2,3, 4, 5, 10, 20, or 25 amino acids, provided that such sequence still iscapable of providing any of the properties of LEs disclosed herein.

In some embodiments, the lymphoproliferative element is a chimericcytokine receptor such as but not limited to a cytokine tethered to itsreceptor that typically constitutively activates the same STAT pathwayas a corresponding activated wild-type cytokine receptor such as STAT3,STAT4, and in illustrative embodiments, STAT5. In some embodiments, thechimeric cytokine receptor is an interleukin, or a fragment thereof,tethered to or covalently attached to its cognate receptor, or afragment thereof, via a linker. In some embodiments, the chimericcytokine receptor is IL7 tethered to IL7Rα (also known as IL7RA). Inother embodiments, the chimeric cytokine receptor is IL-7 tethered to adomain of IL7Rα, such as for example, the extracellular domain of IL-7Rαand/or the transmembrane domain of IL-7Rα. In some embodiments, thelymphoproliferative element is a cytokine receptor that is not tetheredto a cytokine, and in fact in illustrative embodiments, provided hereina lymphoproliferative element is a constitutively active cytokinereceptor that is not tethered to a cytokine. These chimeric IL-7receptors typically constitutively activate STAT5 when expressed.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, wherein thelymphoproliferative element is a cytokine or cytokine receptorpolypeptide, or a fragment thereof comprising a signaling domain, thelymphoproliferative element can comprise an interleukin polypeptidecovalently attached to a portion of its cognate interleukin receptorpolypeptide via a linker. Typically, this portion of the cognateinterleukin receptor includes a functional portion of the extracellulardomain capable of binding the interleukin cytokine and the transmembranedomain. In some embodiments, the intracellular domain is anintracellular portion of the cognate interleukin receptor. In someembodiments, the intracellular domain is an intracellular portion of adifferent cytokine receptor that is capable of promoting lymphocyteproliferation. In some embodiments the lymphoproliferative element is aninterleukin polypeptide covalently attached to its full length cognateinterleukin receptor polypeptide via a linker.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the protein IL7RA.The domains, motifs, and point mutations of IL7RA that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL7RA polypeptides, some of which arediscussed in this paragraph. The IL7RA protein has an S region rich inserine residues (359-394 of full-length IL7RA, corresponding to residues96-133 of SEQ ID NO:248), a T region with three tyrosine residues(residues Y401, Y449, and Y456 of full-length IL7RA, corresponding toresidues Y138, Y18, and Y193 of SEQ ID NO:248), and a Box1 motif thatcan bind the signaling kinase Jak1 (residues 272-280 of full-lengthIL7RA corresponding to residues 9-17 of SEQ ID NO:248 and 249) (Jiang,Qiong et al. Mol. and Cell. Biol. Vol. 24(14):6501-13 (2004)). In someembodiments, a lymphoproliferative element herein can include one ormore, for example all of the domains and motifs of IL7RA disclosedherein or otherwise known to induce proliferation and/or survival of Tcells and/or NK cells. In some embodiments, a suitable intracellulardomain can include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch ofat least 10, 15, 20, or all of tire amino acids in SEQ ID NOs:248 or249. In some embodiments, the intracellular domain derived from IL7RAhas a length of from about 30 aa to about 35 aa, from about 35 aa toabout 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa,from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, fromabout 70 aa to about 100 aa, from about 100 aa to about 125 aa, fromabout 125 aa to 150 aa, from about 150 to about 175 aa, or from about175 aa to about 200 aa. In illustrative embodiments, the intracellulardomain derived from IL7RA has a length of from about 30 aa to about 200aa. In illustrative embodiments of lymphoproliferative elements thatinclude a first intracellular domain derived from IL7RA, the secondintracellular domain can be derived from TNFRSF8.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the proteinIL12RB. The domains, motifs, and point mutations of IL12RB that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL12RB polypeptides, some of which arediscussed in this paragraph Full-length IL12RB contains at least oneBox1 motif PXXP (SEQ ID NO:306) where each X can be any amino acid(residues 10-12 of SEQ ID NOs:254 and 255; and residues 107-110 and139-142 of SEQ ID NO:256) (Presky D H et al. Proc Natl Acad Sci USA.1996 Nov. 26; 93(24)). In some embodiments, a lymphoproliferativeelement that includes an IL12RB intracellular domain can include one ormore of the above Box1 motifs or other motifs, domains, or mutations ofIL12RB known to induce proliferation and/or survival of T cells and/orNK cells. The Box1 motifs of IL12RB are known in the art and a skilledartisan can identify corresponding motifs in IL12RB polypeptides. Insome embodiments, a suitable intracellular domain can include a domainwith at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to a stretch of at least 10, 15, 20, or all ofthe amino acids in SEQ ID NOs:254-256. In some embodiments, theintracellular domain derived from IL12RB has a length of from about 30aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa toabout 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa,from about 65 aa to about 70 aa, from about 70 aa to about 100 aa, fromabout 100 aa to about 125 aa, from about 125 aa to 150 aa, from about150 to about 175 aa, from about 175 aa to about 200 aa, or from about200 aa to about 219 aa. In illustrative embodiments, the intracellulardomain derived from IL12RB has a length of from about 30 aa to about 219aa, for example, 30 aa to 92 aa, or 30 aa to 90 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the proteinIL31RA. The domains, motifs, and point mutations of IL31RA that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL31RA polypeptides, some of which arediscussed in this paragraph. Full-length IL31RA contains the Box1 motifPXXP (SEQ ID NO:306) where each X can be any amino acid (correspondingto residues 12-15 of SEQ ID NOs:275 and 276) (Comelissen C et al. Eur JCell Biol. 2012 June-July; 91(6-7):552-66). In some embodiments, alymphoproliferative element that includes an IL31RA intracellular domaincan include tire Box1 motif. Full-length IL31RA also contains threephosphorylatable tyrosine residues that are important for downstreamsignaling, Y652, Y683, and Y721 (corresponding to residues Y96, Y237,and Y165 of SEQ ID NO:275; these tyrosine residues are not present inSEQ ID NO:276) (Comelissen C et al. Eur J Cell Biol. 2012 June-July;91(6-7):552-66). All three tyrosine residues contribute to theactivation of STAT1, while Y652 is required for STAT5 activation andY721 recruits STAT3. In some embodiments, a lymphoproliferative elementwith an IL31RA intracellular domain includes the Box1 motif and/or theknown phosphorylation sites disclosed herein. The Box1 motif andphosphorylatable tyrosines of IL31RA are known in the art and a skilledartisan will be able to identify corresponding motifs andphosphorylatable tyrosines in similar IL31RA polypeptides. In otherembodiments, a lymphoproliferative element with an IL31RA intracellulardomain does not include the known phosphorylation sites disclosedherein. In some embodiments, a suitable intracellular domain can includea domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NOs:275 or 276. In some embodiments,the intracellular domain derived from IL31RA has a length of from about30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aato about 45 aa, from about 45 aa to about 50 aa, from about 50 aa toabout 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 100aa, from about 100 aa to about 125 aa, from about 125 aa to 150 aa, fromabout 150 to about 175 aa, or from about 175 aa to about 189 aa. Inillustrative embodiments, the intracellular domain derived from IL31RAhas a length of from about 30 aa to about 200 aa, for example, 30 aa to189 aa, 30 aa to 106 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion of thetransmembrane protein CD40. The domains, motifs, and point mutations ofCD40 that induce proliferation and/or survival of T cells and/or NKcells are known in the art and a skilled artisan can identifycorresponding domains, motifs, and point mutations in CD40 polypeptides,some of which are discussed in this paragraph. The CD40 protein containsseveral binding sites for TRAP proteins. Not to be limited by theory,binding sites for TRAF1, TRAF2, and TRAF3 are located at the membranedistal domain of the intracellular portion of CD40 and include the aminoacid sequence PXQXT (SEQ ID NO:303) where each X can be any amino acid,(corresponding to amino acids 35-39 of SEQ ID NO:208) (Elgueta et al.Immunol Rev. 2009 May; 229(1):152-72). TRAF2 has also been shown to bindto the consensus sequence SXXE (SEQ ID NO:304) where each X can be anyamino acid, (corresponding to amino acids 57-60 of SEQ ID NO:208)(Elgueta et al. Immunol Rev. 2009 May; 229(1):152-72). A distinctbinding site for TRAF6 is situated at the membrane proximal domain ofintracellular portion of CD40 and includes the consensus sequence QXPXEX(SEQ ID NO:305) where each X can be any amino acid (corresponding toamino acids 16-21 of SEQ ID NO:208) (Lu et al. J Biol Chem. 2003 Nov.14; 278(46):45414-8). In illustrative embodiments, the intracellularportion of the transmembrane protein CD40 can include all the bindingsites for the TRAP proteins. The TRAP binding sites are known in the artand a skilled artisan will be able to identify corresponding TRAPbinding sites in similar CD40 polypeptides. In some embodiments, asuitable intracellular domain can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all of the amino acidsin SEQ ID NO:208 or SEQ ID NO:209. In some embodiments, theintracellular domain derived from CD40 has a length of from about 30amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60aa to about 65 aa. In illustrative embodiments, the intracellular domainderived from CD40 has a length of from about 30 aa to about 66 aa, forexample, 30 aa to 65 aa, or 50 aa to 66 aa. In illustrative embodimentsof lymphoproliferative elements that include a first intracellulardomain derived from CD40, the second intracellular domain can be otherthan an intracellular domain derived from MyD88, a CD28 family member(e.g. CD28, ICOS), Pattern Recognition Receptor, a C-reactive proteinreceptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor, CD40,RANK/TRANCE-R, OX40, 4-1BB), an HSP receptor (Lox-1 and CD91), or CD28.Pattern Recognition Receptors include, but are not limited to endocyticpattern-recognition receptors (i.e., mannose receptors, scavengerreceptors (i.e., Mac-1, LRP, peptidoglycan, techoic acids, toxins, CD1 1c/CR4)); external signal pattern-recognition receptors (Toll-likereceptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10),peptidoglycan recognition protein, (PGRPs bind bacterial peptidoglycan,and CD14); internal signal pattern-recognition receptors (i.e.,NOD-receptors 1 & 2), and RIG1

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofCD27. The domains, motifs, and point mutations of CD27 that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in CD27 polypeptides, some of which arediscussed in this paragraph. The serine at amino acid 219 of full-lengthCD27 (corresponding to the serine at amino acid 6 of SEQ ID NO:205) hasbeen shown to be phosphorylated. In some embodiments, a suitableintracellular domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 91%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:205. In some embodiments, the intracellular domain derived from CD27has a length of from about 30 amino acids (aa) to about 35 aa, fromabout 35 aa to about 40 aa, from about 40 aa to about 45 aa, or fromabout 45 aa to about 50 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofCSF2RB. The domains, motifs, and point mutations of CSF2RB that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in CSF2RB polypeptides, some of which arediscussed in this paragraph. Full-length CSF2RB contains a Box1 motif atamino acids 474-482 (corresponding to amino acids 14-22 of SEQ IDNO:213). The tyrosine at amino acid 766 of full-length CSF2RB(corresponding to the tyrosine at amino acid 306 of SEQ ID NO: 213) hasbeen shown to be phosphorylated. In some embodiments, a suitableintracellular domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96% 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO: 213. In some embodiments, the intracellular domain derived fromCSF2RB has a length of from about 30 aa to about 35 aa, from about 35 aato about 40 aa, from about 40 aa to about 45 aa, from about 45 aa toabout 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa,from about 70 aa to about 100 aa, from about 100 aa to about 125 aa,from about 125 aa to 150 aa, from about 150 to about 175 aa, from about175 aa to about 200 aa, from about 200 aa to about 250 aa, from about250 aa to 300 aa, from about 300 aa to 350 aa, from about 350 aa toabout 400 aa, or from about 400 aa to about 450 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIL2RB. The domains, motifs, and point mutations of IL2RB that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL2RB polypeptides, some of which arediscussed in this paragraph. Full-length IL2RB contains a Box1 motif atamino acids 278-286 (corresponding to amino acids 13-21 of SEQ IDNO:240). In some embodiments, a suitable intracellular domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:240. In someembodiments, the intracellular domain derived from IL2RB has a length offrom about 30 aa to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aato about 65 aa, from about 65 aa to about 70 aa, from about 70 aa toabout 100 aa, from about 100 aa to about 125 aa, from about 125 aa to150 aa, from about 150 to about 175 aa, from about 175 aa to about 200aa, from about 200 aa to about 250 aa, or from about 250 aa to 300 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIL6ST. The domains, motifs, and point mutations of IL6ST that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL6ST polypeptides, some of which arediscussed in this paragraph. Full-length IL6ST contains a Box1 motif atamino acids 651-659 (corresponding to amino acids 10-18 of SEQ IDNO:247). The serines at amino acids 661, 667, 782, 789, 829, and 839 offull-length IL6ST (corresponding to serines at amino acids 20, 26, 141,148, 188, and 198, respectively, of SEQ ID NO:247) have been shown to bephosphorylated. In some embodiments, a suitable intracellular domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identify to a stretch of at least10, 15, 20, or all of tire amino acids in SEQ ID NO:246 or SEQ IDNO:247. In some embodiments, the intracellular domain derived from IL6SThas a length of from about 30 aa to about 35 aa, from about 35 aa toabout 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa,from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, fromabout 70 aa to about 100 aa, from about 100 aa to about 125 aa, fromabout 125 aa to 150 aa, from about 150 to about 175 aa, from about 175aa to about 200 aa, from about 200 aa to about 250 aa, or from about 250aa to 300 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIL17RE. The domains, motifs, and point mutations of IL17RE that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL17RE polypeptides, some of which arediscussed in this paragraph. Full-length IL17RE contains a TIR domain atamino acids 372-495 (corresponding to amino acids 13-136 of SEQ IDNO:265). In some embodiments, a suitable intracellular domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identify to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:265. In someembodiments, the intracellular domain derived from IL17RE has a lengthof from about 30 aa to about 35 aa, from about 35 aa to about 40 aa,from about 40 aa to about 45 aa, from about 45 aa to about 50 aa fromabout 50 aa to about 55 aa, from about 55 aa to about 60 aa from about60 aa to about 65 aa from about 65 aa to about 70 aa from about 70 aa toabout 100 aa from about 100 aa to about 125 aa from about 125 aa to 150aa from about 150 to about 175 aa or from about 175 aa to about 200 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIL2RG. The domains, motifs, and point mutations of IL2RG that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL2RG polypeptides, some of which arediscussed in this paragraph. Full-length IL2RG contains a Box1 motif atamino acids 286-294 (corresponding to amino acids 3-11 of SEQ IDNO:241). In some embodiments, a suitable intracellular domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:241. In someembodiments, the intracellular domain derived from IL2RG has a length offrom about 30 aa to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aato about 65 aa, from about 65 aa to about 70 aa, or from about 70 aa toabout 100 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIL18R1. The domains, motifs, and point mutations of IL18R1 that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL18R1 polypeptides, some of which arediscussed in this paragraph. Full-length IL18R1 contains a TIR domain atamino acids 222-364 (corresponding to amino acids 28-170 of SEQ IDNO:266). In some embodiments, a suitable intracellular domain caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:266. In someembodiments, the intracellular domain derived from IL18R1 has a lengthof from about 30 aa to about 35 aa, from about 35 aa to about 40 aa,from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, fromabout 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about60 aa to about 65 aa, from about 65 aa to about 70 aa, or from about 70aa to about 100 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIL27RA. The domains, motifs, and point mutations of IL27RA that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IL27RA polypeptides, some of which arediscussed in this paragraph. Full-length IL27RA contains a Box1 motif atamino acids 554-562 (corresponding to amino acids 17-25 of SEQ IDNO:273). In some embodiments, a suitable intracellular domain caninclude a domain with at least 50% 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:273 or SEQ ID NO:274.In some embodiments, the intracellular domain derived from IL27RA has alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, from about 65 aa to about 70 aa, or fromabout 70 aa to about 100 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from an intracellular portion ofIFNGR2. The domains, motifs, and point mutations of IFNGR2 that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in IFNGR2 polypeptides, some of which arediscussed in this paragraph. Full-length IFNGR2 contains a dileucineinternalization motif at amino acids 276-277 (corresponding to aminoacids 8-9 of SEQ ID NO:230). In some embodiments, a suitableintracellular domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identify toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO:230. In some embodiments, the intracellular domain derived fromIFNGR2 has a length of from about 30 aa to about 35 aa, from about 35 aato about 40 aa, from about 40 aa to about 45 aa, from about 45 aa toabout 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the protein MyD88.The domains, motifs, and point mutations of MyD88 that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in MyD88 polypeptides, some of which arediscussed in this paragraph. The MyD88 protein has an N-terminal deathdomain that mediates interactions with other death domain-containingproteins (corresponding to amino acids 29-106 of SEQ ID NO:284), anintermediate domain that interacts with IL-1R associated kinase(corresponding to amino acids 107-156 of SEQ ID NO:284), and aC-terminal T1R domain (corresponding to amino acids 160-304 of SEQ IDNO:284) that associates with the TLR-TIR domain (Biol Res. 2007;40(2):97-112). MyD88 also has canonical nuclear localization and exportmotifs. Point mutations have been identified in MyD88 and include theloss-of-function mutations L93P and R193C (corresponding to L93P andR196C in SEQ ID NO:284), and the gain-of-function mutation L265P(corresponding to L260P in SEQ ID NO:284) (Deguine and Barton.F1000Prime Rep. 2014 Nov. 4; 6:97). In some embodiments, alymophoproliferative element herein can include one or mote, for exampleall of the domains and motifs of MyD88 disclosed herein. In someembodiments, a suitable intracellular domain can include a domain withat least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to a stretch of at least 10, 15, 20, or all ofthe amino acids in SEQ ID NO:284-293, and in illustrative embodimentsincludes one or more, in illustrative embodiments all, of the followingMyD88 domains/motifs: the death domain, the intermediate domain, the TIRdomain, the nuclear localization and export motifs, an amino acidcorresponding to position L93, R193, and L265 or P265. In someembodiments, the intracellular domain derived from MyD88 has a length offrom about 30 aa to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aato about 65 aa, from about 65 aa to about 70 aa, from about 70 aa toabout 100 aa, from about 100 aa to about 125 aa, from about 125 aa to150 aa, from about 150 to about 175 aa, from about 175 aa to about 200aa, from about 200 aa to about 250 aa, from about 250 aa to 300 aa, orfrom about 300 aa to 350 aa. In illustrative embodiments, theintracellular domain derived from MyD88 has a length of from about 30 aato about 350 aa, for example, 50 aa to 350 aa, or 100 aa to 350 aa, 100aa to 304 aa, 100 aa to 296 aa, 100 aa to 251 aa, 100 aa to 191 aa, 100aa to 172 aa, 100 aa to 146 aa, or 100 aa to 127 aa. In illustrativeembodiments of lymphoproliferative elements that include a firstintracellular domain derived from MyD88, the second intracellular domaincan be derived from TNFRSF4 or TNFRSF8. In other illustrativeembodiments of lymphoproliferative elements that include a firstintracellular domain derived from MyD88, the second intracellular domaincan be other than an intracellular domain derived from a CD28 familymember (e.g. CD28, ICOS), Pattern Recognition Receptor, a C-reactiveprotein receptor (i.e., Nodi, Nod2, PtX3-R), a INF receptor (i.e., CD40,RANK/TRANCE-R, OX40, 4-1BB), an HSP receptor (Lox-1 and CD91), or CD28.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the transmembraneprotein MPL. The domains, motifs, and point mutations of MPL that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in MPL polypeptides, some of which arediscussed in this paragraph. The transmembrane MPL protein contains theBox1 motif PXXP (SEQ ID NO:306) where each X can be any amino acid(corresponding to amino acids 17-20 in SEQ ID NO:283) and the Box2motif, a region with increased serine and glutamic acid content(corresponding to amino acids 46-64 in SEQ ID NO:283) (Drachman andKaushansky. Proc Natl Acad Sci USA. 1997 Mar. 18; 94(6):2350-5). TheBox1 and Box2 motifs are involved in binding to JAKs and signaltransduction, although the Box2 motif presence is not always requiredfor a proliferative signal (Murakami et al. Proc Natl Acad Sci USA. 1991Dec. 15; 88(24):11349-53; Fukunaga et al. EMBO J. 1991 October;10(10):2855-65; and O'Neal and Lee. Lymphokine Cytokine Res. 1993October; 12(5):309-12). Many cytokine receptors have hydrophobicresidues at positions −1, −2, and −6 relative to the Box1 motif(corresponding to amino acids 16, 15, and 11, respectively, of SEQ IDNO:283), that form a “switch motif,” which is required forcytokine-induced JAK2 activation but not for JAK2 binding(Constantinescu et al. Mol Cell. 2001 February; 7(2):377-85; and Huanget al. Mol Cell. 2001 December; 8(6):1327-38). Deletion of the regionencompassing amino acids 70-95 in SEQ ID NO:283was shown to supportviral transformation in the context of v-mpl (Benit et al. J Virol. 1994August; 68(8):5270-4), thus indicating that this region is not necessaryfor the function of mpl in this context. Morello et al. Blood 1995 July;86(8):557-71 used the same deletion to show that this region was notrequired for stimulating transcription for a hematopoietinreceptor-responsive CAT reporter gene construct and furthermore saw thatthis deletion resulted in slightly enhanced transcription expected forremoval of a nonessential and negative element in this region assuggested by Drachman and Kaushansky. Thus, in some embodiments, a MPLintracellular signaling domain does not comprise the region comprisingamino acids 70-95 in SEQ ID NO:283. In full-length MPL, the lysines K553(corresponding to K40 of SEQ ID NO: 283) and K573 (corresponding to K60of SEQ ID NO: 283) have been shown to be negative regulatory sites thatfunction as part of a ubiquitination targeting motif (Saur et al. Blood2010 Feb. 11; 115(6):1254-63). Thus, in some embodiments herein, a MPLintracellular signaling domain does not comprise these ubiquitinationtargeting motif residues. In full-length MPL, the tyrosines Y521(corresponding to Y8 of SEQ ID NO: 283), Y542 (corresponding to Y29 ofSEQ ID NO:283), Y591 (corresponding to Y78 of SEQ ID NO: 283), Y626(corresponding to Y113 of SEQ ID NO: 283), and Y631 (corresponding toY118 of SEQ ID NO: 283) have been shown to be phosphorylated (Vargheseet al. Front Endocrinol (Lausanne). 2017 Mar. 31; 8:59). Y521 and Y591of full-length MPL are negative regulatory sites that function either aspart of a lysosomal targeting motif (Y521) or via an interaction withadaptor protein AP2 (Y591) (Drachman and Kaushansky. Proc Natl Acad SciUSA. 1997 Mar. 18; 94(6):2350-5; and Hitchcock et al. Blood. 2008 Sep.15; 112(6):2222-31). Y626 and Y631 of full-length MPL are positiveregulatory sites (Drachman and Kaushansky. Proc Natl Acad Sci USA. 1997Mar. 18; 94(6):2350-5) and the murine homolog of Y626 is required forcellular differentiation and the phosphorylation of Shc (Alexander etal. EMBO J. 1996 Dec. 2; 15(23):6531-40) and Y626 is also required forconstitutive signaling in MPL with the W515A mutation described below(Pecquet et al. Blood. 2010 Feb. 4; 115(5):1037-48). MPL contains theShc phosphotyrosine-binding binding motif NXXY (SEQ ID NO:307) whereeach X can be any amino acid (corresponding to amino acids 110-113 ofSEQ ID NO: 283), and this tyrosine is phosphorylated and important forthe TPO-dependent phosphorylation of She, SHIP, and STAT3 (Laminet etal. J Biol Chem. 1996 Jan. 5; 271(1):264-9; and van der Geer et al. ProcNatl Acad Sci USA. 1996 Feb. 6; 93(3):963-8). MPL also contains theSTAT3 consensus binding sequence YXXQ (SEQ ID NO:308) where each X canbe any amino acid (corresponding to amino acids 118-121 of SEQ ID NO:283) (Stahl et al. Science. 1995 Mar. 3; 267(5202):1349-53). Thetyrosine of this sequence can be phosphorylated and MPL is capable ofpartial STAT3 recruitment (Drachman and Kaushansky. Proc Natl Acad SciUSA. 1997 Mar. 18; 94(6):2350-5). MPL also contains the sequence YLPL(SEQ ID NO: 309) (corresponding to amino acid 113-116 of SEQ ID NO:283), which is similar to the consensus binding site for STAT5recruitment pYLXL (SEQ ID NO:310) where pY is phosphotyrosine and X canbe any amino acid (May et al. FEBS Lett. 1996 Sep. 30; 394(2):221-6).Using computer simulations, Lee et al. found clinically relevantmutations in the transmembrane domain of MPL should activate MPL withthe following order of activating effects: W515K (corresponding to theamino acid substitution W2K of SEQ ID NO: 283)>S505A (corresponding tothe amino acid substitution S14A of SEQ ID NO:187)>W515I (correspondingto the amino acid substitution W2I of SEQ ID NO: 283)>S505N(corresponding to the amino acid substitution S14N of SEQ ID NO:187,which was tested in Example 12 as part T075 (SEQ ID NO:188)) (PLoS One.2011; 6(8):e23396). The simulations predicted these mutations couldcause constitutive activation of JAK2, the kinase partner of MPL. Insome embodiments, the intracellular portion of MPL can include one ormore, or all the domains and motifs described herein that are present inSEQ ID NO: 283. In some embodiments, a transmembrane portion of MPL caninclude one or more, or all tire domains and motifs described hereinthat are present in SEQ ID NO:187. The domains, motifs, and pointmutations of MPL provided herein are known in the art and a skilledartisan would recognize that MPL intracellular signaling domains hereinin illustrative embodiments would include one or more correspondingdomains, motifs, and point mutations in that have been shown to promoteproliferative activity and would not include that that have been shownto inhibit MPLs proliferative activity. In some embodiments, a suitableintracellular domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO: 283. In some embodiments, the intracellular domain derived from MPLhas a length of from about 30 aa to about 35 aa, from about 35 aa toabout 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa,from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, fromabout 70 aa to about 100 aa from about 100 aa to about 125 aa from about125 aa to 150 aa from about 150 to about 175 aa from about 175 aa toabout 200 aa from about 200 aa to about 250 aa from about 250 aa to 300aa from about 300 aa to 350 aa from about 350 aa to about 400 aa fromabout 400 aa to about 450 aa from about 450 aa to about 500 aa fromabout 500 aa to about 550 aa from about 550 aa to about 600 aa or fromabout 600 aa to about 635 aa. In illustrative embodiments, theintracellular domain derived from MPL has a length of from about 30 aato about 200 aa for example, 30 aa to 150 aa 30 aa to 119 aa 30 aa to121 aa 30 aa to 122 aa or 50 aa to 125 aa. In illustrative embodimentsof lymphoproliferative elements that include a first intracellulardomain derived from MPL, the second intracellular domain can be derivedfrom CD79B.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element theintracellular domain can be derived from a portion of the transmembraneprotein CD79B, also known as B29; IGB; AGM6. The domains, motifs, andpoint mutations of CD79B that induce proliferation and/or survival of Tcells and/or NK cells are known in the art and a skilled artisan canidentify corresponding domains, motifs, and point mutations in CD79Bpolypeptides, some of which are discussed in this paragraph. CD79Bcontains an ITAM motif at residues 193-212 (corresponding to amino acids16-30 of SEQ ID NO:211). CD79B has two tyrosines that are known to bephosphorylated, Y196 and Y207 (corresponding to Y16 and Y27 of SEQ IDNO: 211). In some embodiments, the intracellular portion of thetransmembrane protein CD79B includes the ITAM motif and/or the knownphosphorylation sites disclosed herein. The motif and phosphorylatabletyrosines of CD79B are known in the art and a skilled artisan will beable to identify corresponding motifs and phosphorylatable tyrosines insimilar CD79B polypeptides. In some embodiments, a suitableintracellular domain can include a domain with at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa stretch of at least 10, 15, 20, or all of the amino acids in SEQ IDNO: 211. In some embodiments, the intracellular domain derived fromCD79B has a length of from about 30 aa to about 35 aa, from about 35 aato about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa toabout 50 aa.). In illustrative embodiments, the intracellular domainderived from CD79B has a length of from about 30 aa to about 50 aa. Forexample, a suitable CD79B intracellular activating domain can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all amino acids of the following sequence:LDKDDSKAGMEEDHT[YEGLDIDQTATYEDI]VTLRTGEVKWSVGEHPGQE (SEQ ID NO: 211),where the ITAM motif is set out in brackets. In illustrative embodimentsof lymphoproliferative elements that include a second intracellulardomain derived from CD79B, the first intracellular domain can be derivedfrom CSF3R.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element theintracellular domain can be derived from a portion of the transmembraneprotein OSMR. The domains, motifs, and point mutations of OSMR thatinduce proliferation and/or survival of T cells and/or NK cells areknown in the art and a skilled artisan can identify correspondingdomains, motifs, and point mutations in OSMR polypeptides, some of whichare discussed in this paragraph. OSMR contains a Box1 motif at aminoacids 771-779 of isoform 3 (corresponding to amino acids 16-30 of SEQ IDNO:294). OSMR has two serines at amino acids 829 and 890 of isoform 3that are known to be phosphorylated (serines at amino acids 65 and 128of SEQ ID NO:294). In some embodiments, the intracellular portion of theprotein OSMR can include the Box1 motif and the known phosphorylationsites disclosed herein. The motif and phosphorylatable serines of OSMRare known in the art and a skilled artisan will be able to identifycorresponding motifs and phosphorylatable serines in similar OSMRpolypeptides. In some embodiments, a suitable intracellular domain caninclude a domain with at least 50% 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identify to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:294. In someembodiments, the intracellular domain derived from OSMR has a length offrom about 30 aa to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aato about 65 aa, from about 65 aa to about 70 aa, from about 70 aa toabout 100 aa, from about 100 aa to about 125 aa, from about 125 aa to150 aa, from about 150 to about 175 aa, from about 175 aa to about 200aa, or from about 200 aa to about 250 aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the transmembraneprotein PRLR. The domains, motifs, and point mutations of PRLR thatinduce proliferation and/or survival of T cells and/or NK cells areknown in the art and a skilled artisan can identify correspondingdomains, motifs, and point mutations in PRLR polypeptides, some of whichare discussed in this paragraph. PRLR contains a growth hormone receptorbinding domain at amino acids 185-261 of isoform 6 (corresponding toamino acids 28-104 of SEQ ID NO:295). The growth hormone receptorbinding domain of PRLR is known in the art and a skilled artisan will beable to identify corresponding domain in similar PRLR polypeptides. Insome embodiments, a suitable intracellular domain can include a domainwith at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to a stretch of at least 10, 15, 20, or all ofthe amino acids in SEQ ID NO:295. In some embodiments, the intracellulardomain derived from PRLR has a length of from about 30 aa to about 35aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about175 aa, from about 175 aa to about 200 aa, from about 200 aa to about250 aa, from about 250 aa to 300 aa, from about 300 aa to 350 aa, orfrom about 350 aa to about 400 aa.

In some embodiments, an intracellular domain of a lymphoproliferativeelement is derived from an intracellular portion of the transmembraneprotein CD30 (also known as TNFRSF8, DlS166E, and Ki-1).

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the protein CD28.The domains, motifs, and point mutations of CD28 that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in CD28 polypeptides, some of which arediscussed in this paragraph. Full-length CD28 contains a PI3-K- andGrb2-binding motif that corresponds to residues 12-15 of SEQ ID NOs:206and 207 (Harada et al. J Exp Med. 2003 Jan. 20; 197(2):257-62). In someembodiments, a lymphoproliferative element that includes a CD28intracellular domain can include the PI3-K- and Grb2-binding motif. Insome embodiments, a suitable intracellular domain can include a domainwith at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identify to a stretch of at least 10, 15, 20, or all ofthe amino acids in SEQ ID NOs:206 or 207. In some embodiments, theintracellular domain derived from CD28 has a length of from about 5 aato about 10 aa, from about 10 aa to about 15 aa, from about 15 aa toabout 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about30 aa, from about 30 aa to about 35 aa, or from about 35 aa to about 42aa.

In illustrative embodiments of any of the methods and compositionsprovided herein that include a lymphoproliferative element, theintracellular domain can be derived from a portion of the protein ICOS.The domains, motifs, and point mutations of ICOS that induceproliferation and/or survival of T cells and/or NK cells are known inthe art and a skilled artisan can identify corresponding domains,motifs, and point mutations in ICOS polypeptides, some of which arediscussed in this paragraph. Unlike CD28, ICOS binds PI3-K and not Grb2.The PI3-K-binding motif of full-length ICOS corresponds to residues19-22 of SEQ ID NO:225. A single amino acid substitution in this motifcan lead to Grb2 binding by ICOS and increased IL-2 production (Haradaet al. J Exp Med. 2003 Jan. 20; 197(2):257-62). This mutationcorresponds to mutating phenylalanine 21 of SEQ ID NO:225 to anasparagine. A skilled artisan will understand how to mutate this residuein SEQ ID NO:225 and generate an ICOS intracellular domain that bindsGrb2 in addition to PI3-K. In some embodiments, a lymphoproliferativeelement that includes an ICOS intracellular domain can include thePI3-K-binding motif. In some embodiments, a lymphoproliferative elementthat includes an ICOS intracellular domain can include the PI3-K-bindingmotif that has been mutated to additionally bind Grb2. ICOS alsocontains a membrane proximal motif in the cytoplasmic tail that isessential for ICOS-assisted calcium signaling (Leconte et al. MolImmunol. 2016 November; 79:38-46). This calcium signaling-motifcorresponds to residues 5-8 of SEQ ID NO:225. In some embodiments, alymphoproliferative element that includes an ICOS intracellular domaincan include the calcium-signaling motif. Two other conserved motifs havebeen identified in full-length ICOS. A first conserved motif at residues170-179 (corresponding to residues 9-18 of SEQ ID NO:225) and a secondconserved motif at residues 185-191 (corresponding to residues 24-30 ofSEQ ID NO:225) (Pedros et al. Nat Immunol. 2016 July; 17(7):825-33).These two conserved motifs might have important function(s) in mediatingdownstream ICOS signaling. In some embodiments, a lymphoproliferativeelement that includes an ICOS intracellular domain can include at leastone of the first or second conserved motifs. In some embodiments, alymphoproliferative element that includes an ICOS intracellular domaindoes not include the first conserved motif, does not include the secondconserved motif, or does not include the first and second conservedmotifs. In some embodiments, a suitable intracellular domain can includea domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NO:225. In some embodiments, theintracellular domain derived from ICOS has a length of from about 5 aato about 10 aa, from about 10 aa to about 15 aa, from about 15 aa toabout 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about30 aa, from about 30 aa to about 35 aa, or from about 35 aa to about 38aa.

In some embodiments, an intracellular domain of a chimericlymphoproliferative element is derived from an intracellular portion ofthe transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3,ACT35, CD134, OX-40, TXGPlL). The domains, motifs, and point mutationsof OX40 that induce proliferation and/or survival of T cells and/or NKcells are known in the art and a skilled artisan can identifycorresponding domains, motifs, and point mutations in OX40 polypeptides,some of which are discussed in this paragraph. OX40 contains a TRAFbinding motif at residues 256-263 of full-length OX40 (corresponding toresidues 20-27 of SEQ ID NO:296) that are important for binding TRAF1,TRAF2, TRAF3, and TRAF5 (Kawamata, S, et al. J Biol Chem. 1998 Mar. 6;273(10):5808-14; Hori, T. Int J Hematol. 2006 January; 83(1):17-22).Full-length OX40 also contains a p85 PI3K binding motif at residues34-57. In some embodiments, when OX40 is present as an intracellulardomain of a lymphoproliferative element, it includes the p85 PI3Kbinding motif of OX40. In some embodiments, an intracellular domain ofOX40 can include the TRAF binding motif of OX40. In some embodiments, anintracellular domain of OX40 can bind TRAF1, TRAF2, TRAF3, and TRAF5.Lysines corresponding to amino acids 17 and 41 of SEQ ID NO: 296 arepotentially negative regulatory sites that function as parts ofubiquitin targeting motifs. In some embodiments, one or both of theselysines in the intracellular domain of OX40 are mutated arginines oranother amino acid. In some embodiments, a suitable intracellular domainof a lymphoproliferative element can include a domain with at least 50%,60%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99% or 100% sequenceidentify to a stretch of at least 10, 15, 20, or all of the amino acidsin SEQ ID NO:57. In some of these embodiments, the intracellular domainof OX40 has a length of from about 20 aa to about 25 aa, about 25 aa toabout 30 aa, 30 aa to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, or from about 45 aa to about 50 aa. Inillustrative embodiments, the intracellular domain of OX40 has a lengthof from about 20 aa to about 50 aa, for example 20 aa to 45 aa, or 20 aato 42 aa.

In some embodiments, an intracellular domain of a chimericlymphoproliferative element is derived from an intracellular portion ofthe transmembrane protein IFNAR2. The domains, motifs, and pointmutations of IFNAR2 that induce proliferation and/or survival of T cellsand/or NK cells are known in the art and a skilled artisan can identifycorresponding domains, motifs, and point mutations in IFNAR2polypeptides, some of which are discussed in this paragraph. Full-lengthIFNAR2 contains a Box1 motif and two Box2 motifs (known as Box2A andBox2B). (Usacheva A et al. J Biol Chem. 2002 Dec. 13; 277(50):48220-6).In some embodiments, a lymphoproliferative element that includes aIFNAR2 intracellular domain can include one or more of the Box1 or Box2motifs. In illustrative embodiments, the IFNAR2 intracellular domain caninclude one or more of the Box1, Box2A, or Box2B motifs. IFNAR2 containsa JAK1-binding site (Gauzzi M C et al. Proc Natl Acad Sci USA. 1997 Oct.28; 94(22):11839-44; Schindler et al. J Biol Chem. 2007 Jul. 13;282(28):20059-63). In some embodiments, a lymphoproliferative elementthat includes a IFNAR2 intracellular domain can include the JAK1-bindingsite. In some embodiments, a suitable intracellular domain of alymphoproliferative element can include a domain with at least 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all of the amino acidsin SEQ ID NOs:227 or 228. In some of these embodiments, theintracellular domain of IFNAR2 has a length of from about 30 aa to about35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about251 aa. In illustrative embodiments, the intracellular domain of OX40has a length of from about 30 aa to about 251 aa, for example 30 aa to67 aa.

In some embodiments, an intracellular domain of a chimericlymphoproliferative element is derived from an intracellular portion ofthe transmembrane protein CSF3R. The domains, motifs, and pointmutations of CSF3R that induce proliferation and/or survival of T cellsand/or NK cells are known in the art and a skilled artisan can identifycorresponding domains, motifs, and point mutations in CSF3Rpolypeptides, some of which are discussed in this paragraph. Full-lengthCSF3R contains a Box1 and Box2 motif as well as a Box3 motif(Nguyen-Jackson H T et al. G-CSF Receptor Structure, Function, andIntracellular Signal Transduction. Twenty Years of G-CSF, (2011)83-105). In some embodiments, a lymphoproliferative element thatincludes a CSF3R intracellular domain can include one or more of theBox1, Box2, or Box3 motifs. CSF3R contains four tyrosine residues, Y704,Y729, Y744, and Y764 in full-length CSF3R, that are important forbinding STAT3 (Y704 and Y744), SOCS3 (Y729), and Grb2 and p21Ras (Y764).In some embodiments, a lymphoproliferative element that includes a CSF3Rintracellular domain can include one, two, three, or all of the tyrosineresidues corresponding to Y704, Y729, Y744, and Y764 of full-lengthCSF3R. CSF3R contains two threonine residues, T615 and T618 infull-length CSF3R, that can increase receptor dimerization and activitywhen mutated to alanine and isoleucine, respectively (T615A and T618I)(Maxson et al. J Biol Chem. 2014 Feb. 28; 289(9):5820-7). In someembodiments, a lymphoproliferative element that includes a CSF3Rintracellular domain can include one or more of the mutationscorresponding to T615A and T618I. In some embodiments, a suitableintracellular domain of a lymphoproliferative element can include adomain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20,or all of the amino acids in SEQ ID NOs:216, 217, or 218. In some ofthese embodiments, the intracellular domain of CSF3R has a length offrom about 30 aa to about 35 aa, from about 35 aa to about 40 aa, fromabout 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aato about 65 aa, from about 65 aa to about 70 aa, from about 70 aa toabout 100 aa, from about 100 aa to about 125 aa, from about 125 aa to150 aa, from about 150 to about 175 aa, from about 175 aa to about 200aa, or from about 200 aa to about 213 aa. In illustrative embodiments,the intracellular domain of CSF3R has a length of from about 30 aa toabout 213 aa, for example from about 30 aa to about 186 or from about 30aa to about 133 aa.

In some embodiments, an intracellular domain of a chimericlymphoproliferative element is derived from an intracellular portion ofthe transmembrane protein EPOR. The domains, motifs, and point mutationsof EPOR that induce proliferation and/or survival of T cells and/or NKcells are known in the art and a skilled artisan can identifycorresponding domains, motifs, and point mutations in EPOR polypeptides,some of which are discussed in this paragraph. EPOR contains a Box1(residues 257-264 of full-length EPOR) and Box2 (residues 303-313 offull-length EPOR) motif (Constantinescu S N. Trends Endocrinol Metab.1999 December; 10(1):18-23). EPOR also contains an extended Box2 motif(residues 329-372) important for binding tyrosine kinase receptor KIT(Constantinescu S N. Trends Endocrinol Metab. 1999 December;10(1):18-23). In some embodiments, a lymphoproliferative element thatincludes an EPOR intracellular domain can include one or more of theBox1, Box2, or extended Box2 motifs. EPOR also contains a short segmentimportant for EPOR internalization (residues 267-276 of full-lengthEPOR). In some embodiments, a lymphoproliferative element that includesan EPOR intracellular domain does not include the internalizationsegment. In some embodiments, a suitable intracellular domain of alymphoproliferative element can include a domain with at least 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a stretch of at least 10, 15, 20, or all of the amino acidsin SEQ ID NOs:219 or 220. In some of these embodiments, theintracellular domain of EPOR has a length of from about 30 aa to about35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about65 aa to about 70 aa, from about 70 aa to about 100 aa, from about 100aa to about 125 aa, from about 125 aa to 150 aa, from about 150 to about175 aa, from about 175 aa to about 200 aa, or from about 200 aa to about235 aa. In illustrative embodiments, the intracellular domain of EPORhas a length of from about 30 aa to about 235 aa.

In some embodiments, an intracellular domain of a chimericlymphoproliferative element is derived from an intracellular portion ofthe transmembrane protein CD3G. The domains, motifs, and point mutationsof CD3G that induce proliferation and/or survival of T cells and/or NKcells are known in the art and a skilled artisan can identifycorresponding domains, motifs, and point mutations in CD3G polypeptides,some of which are discussed in this paragraph. Two serine residues, SI23 and S126 of full-length CD3G have been shown to be phosphorylated inT cells in response to ionomycin (Davies et al. J Biol Chem. 1987 Aug.15; 262(23):10918-21). In some embodiments, a lymphoproliferativeelement that includes a CD3G intracellular domain can include one ormore of the serine residues corresponding to full-length S123 and S126.Furthermore, phosphorylation at S126 but not S123 was shown to berequired for PKC-mediated down-regulation (Dietrich J et al. EMBO J.1994 May 1; 13(9):2156-66). In some embodiments, a lymphoproliferativeelement that includes a CD3G intracellular domain can include the serineresidue corresponding to full-length S123 and not include serine residuecorresponding to full-length S126. In some embodiments, alymphoproliferative element that includes a CD3G intracellular domaincan include a non-phosphorylatable amino acid substitution at the serineresidue corresponding to full-length S126. In illustrative embodiments,the amino acid substitution can be a serine to alanine mutation.Additionally, leucine to alanine mutations of either leucine of adi-leucine motif, L131 and L132 in full-length CD3G, was shown toprevent PKC-mediated down-regulation (Dietrich J et al. EMBO J. 1994 May1; 13(9):2156-66). In some embodiments, a lymphoproliferative elementthat includes a CD3G intracellular domain can include at least one aminoacid substitution at the leucine residues corresponding to L131 or L132of full-length CD3G. In illustrative embodiments, the amino acidsubstitution can be a leucine to alanine mutation. In some embodiments,a suitable intracellular domain of a lymphoproliferative element caninclude a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least10, 15, 20, or all of the amino acids in SEQ ID NO:199. In some of theseembodiments, the intracellular domain of CD3G has a length of from about20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aato about 35 aa, from about 35 aa to about 40 aa, or from about 40 aa toabout 45 aa. In illustrative embodiments, the intracellular domain ofCD3D has a length of from about 30 aa to about 45 aa.

The cytoplasmic domains of TNF receptors (TNFRs), which in illustrativeembodiments can be TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18, canrecruit signaling molecules, including TRAFs (TNF receptor-associatedfactors) and/or “death domain” (DD) molecules. The domains, motifs, andpoint mutations of TNFRs that induce proliferation and/or survival of Tcells and/or NK cells are known in the art and a skilled artisan canidentify corresponding domains, motifs, and point mutations in TNFRpolypeptides, some of which are discussed in this paragraph. In mammals,there are at least six TRAP molecules and a number of nonreceptor DDmolecules. Receptors and adaptor proteins that bind to TRAFs share shortconsensus TRAF-binding motifs that are known in the art (Meads et al. JImmunol. 2010 Aug. 1; 185(3):1606-15). The DD-binding motif is a roughly60 amino acid globular bundle of 6 conserved α-helices that is alsoknown in the art (Locksley R M et al. Cell. 2001 Feb. 23;104(4):487-501). A skilled artisan will be able to identify the TRAF-and/or DD-binding motif in the different TNFR families using, forexample, sequence alignments to known binding motifs. TNFRs can recruitTRADD and TRAF2, resulting in the activation of NF-κB, MAPK, and JNK(Sedger and McDermott. Cytokine Growth Factor Rev. 2014 August;25(4):453-72). In some embodiments, a lymphoproliferative element thatincludes a TNFR intracellular domain can include one or moreTRAF-binding motifs. In some embodiments, a lymphoproliferative elementthat includes a TNFR intracellular domain does not include a DD-bindingmotif, or has one or more DD-binding motifs deleted or mutated withinthe intracellular domain. In some embodiments, a lymphoproliferativeelement that includes a TNFR intracellular domain can recruit TRADDand/or TRAF2. TNFRs also include cysteine-rich domains (CRDs) that areimportant for ligand binding (Locksley R M et al. Cell. 2001 Feb. 23;104(4):487-501). In some embodiments, a lymphoproliferative element thatincludes a TNFR intracellular domain does not include a TNFR CRD.

Lymphoproliferative elements and CLEs that can be included in any of theaspects disclosed herein, can be any of the LEs or CLEs disclosed inWO2019/055946. CLEs were disclosed therein that promoted proliferationin cell culture of PBMCs dial were transduced with lentiviral particlesencoding the CLEs between day 7 and day 21, 28, 35 and/or 42 aftertransduction. Furthermore, CLEs were identified therein, that promotedproliferation in vivo in mice in the presence or absence of an antigenrecognized by a CAR, wherein T cells expressing one of the CLEs and theCAR were introduced into the mice. As exemplified therein, tests and/orcriteria can be used to identify whether any test polypeptide, includingLEs, or test domains of an LE, such as a first intracellular domain, ora second intracellular domain, or both a first and second intracellulardomain, are indeed LEs or effective intracellular domains of LEs, orespecially effective LEs or intracellular domains of LEs. Thus, incertain embodiments, any aspect or other embodiment provided herein thatincludes an LE or a polynucleotide or nucleic acid encoding an LE canrecite that the LE meets, or provides the property of, or is capable ofproviding and/or possesses the property of, any one or more of theidentified tests or criteria for identifying an LE provided herein, orthat a cell genetically modified and/or transduced with a retroviralparticle, such as a lentiviral particle encoding the LE, is capable ofproviding, is adapted for, possesses the property of, and/or is modifiedfor achieving the results of one or more of the recited tests. In oneembodiment, the LE provides, is capable of providing and/or possessesthe property of, (or a cell genetically modified and/or transduced witha retroviral particle encoding the LE is capable of providing, isadapted for, possesses the property of, and/or is modified for) improvedexpansion to pre-activated PBMCs transduced with a lentivirus comprisinga nucleic acid encoding the LE and an anti-CD19 CAR comprising a CD3zeta intracellular activating domain but no co-stimulatory domain,between day 7 and day 21, 28, 35, and/or 42 of in vitro culturingpost-transduction in the absence of exogenously added cytokines,compared to a control retroviral particle, e.g. lentiviral particleunder identical conditions. In some embodiments, a lymphoproliferativeelement test for improved or enhanced survival, expansion, and/orproliferation of cells transduced with a retroviral particle (e.g.lentiviral particle) having a genome encoding a test construct encodinga putative LE (test cells) can be performed based on a comparison tocontrol cells, which can be, for example, either untransduced cells orcells transduced with a control retroviral (e.g. lentiviral) particleidentical to the lentiviral particle comprising the nucleic acidencoding the lymphoproliferative element, but lacking thelymphoproliferative element, or lacking the intracellular domain ordomains of the test polypeptide construct but comprising the sameextracellular domain, if present, and the same transmembrane region ormembrane targeting region of the respective test polypeptide construct.In some embodiments control cells are transduced with a retroviralparticle (e.g. lentiviral particle) having a genome encoding alymphoproliferative element or intracellular domain(s) thereof,identified herein as exemplifying a lymphoproliferative element. In suchan embodiment, the test criteria can include that there is at least asmuch enrichment survival and/or expansion, or no statistical differenceof enrichment, survival, and/or expansion when the test is performedusing a retroviral particle (e.g. lentiviral particle) having a genomeencoding a test construct versus encoding the controllymphoproliferative element typically by analyzing cells transcribedtherewith. Exemplary or illustrative embodiments of lymphoproliferativeelements herein, in some embodiments, are illustrative embodiments ofcontrol lymphoproliferative elements for such a test.

In some embodiments, this test for an improved property of a putative ortest lymphoproliferative element is performed by performing replicatesand/or performing a statistical test. A skilled artisan will recognizethat many statistical tests can be used for such a lymphoproliferativeelement test. Contemplated for such a test in these embodiments would beany such test known in the art. In some embodiments, the statisticaltest can be a T-test or a Mann-Whitnev-Wilcoxon test. In someembodiments, the normalized enrichment level of a test construct issignificant at a p-value of less than 0.1, or less than 0.05, or lessthan 0.01.

In another embodiment, the LE provides, is capable of providing and/orpossesses the property of (or a cell genetically modified and/ortransduced with the LE is capable of providing, is adapted for,possesses the property of, and/or is modified for) at least a 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold expansion, or between 1.5 fold and 25-fold expansion, or between2-fold and 20-fold expansion, or between 2-fold and 15-fold expansion,or between 5-fold and 25-fold expansion, or between 5-fold and 20-foldexpansion, or between 5-fold and 15-fold expansion, of pre-activatedPBMCs transduced with a nucleic acid encoding the LE when transducedalong with an anti-CD19 CAR comprising a CD3 zeta intracellularactivating domain but no co-stimulatory domain, between day 7 and day21, 28, 35, and/or 42 of in vitro culturing in the absence ofexogenously added cytokines. In some embodiments, the test is performedin the presence of PBMCs, for example at a 1:1 ratio of transduced cellsto PBMCs, which can be for example, from a matched donor, and in someembodiments, the test is performed in the absence of PBMCs. In someembodiments, the analysis of expansion for any of these tests isperformed as illustrated in WO2019/055946. In some embodiments, the testcan include a further statistical test and a cut-off such as a P valuebelow 0.1, 0.05, or 0.01, wherein a test polypeptide or nucleic acidencoding the same, needs to meet one or both thresholds (i.e. foldexpansion and statistical cutoff).

For any of the lymphoproliferative element tests provided herein, thenumber of test cells and the number of control cells can be comparedbetween day 7 and day 14, 21, 28, 35, 42 or 60 post-transduction. Insome embodiments, the numbers of test and control cells can bedetermined by sequencing DNA and counting the occurrences of uniqueidentifiers present in each construct. In some embodiments, the numbersof test and control cells can be counted directly, for example with ahemocytometer or a cell counter. In some embodiments, all the test cellsand control cells can be grown within the same vessel, well or flask. Insome embodiments, the test cells can be seeded in one or more wells,flasks or vessels, and the control cells can be seeded in one or moreflasks or vessels. In some embodiments, test and control cells can beseeded individually into wells or flasks, e.g., one cell per well. Insome embodiments, the numbers of test cells and control cells can becompared using enrichment levels. In some embodiments, the enrichmentlevel for a test or control construct can be calculated by dividing thenumber of cells at a later time point (day 14, 21, 28, 35, or day 45) bythe number of cells at day 7 for each construct. In some embodiments,the enrichment level for a test or control construct can be calculatedby dividing the number of cells at a time point (day 14, 21, 28, 35, orday 45) by the number of cells at that time point for untransducedcells. In some embodiments, the enrichment level of each test constructcan be normalized to the enrichment level of the respective controlconstruct to generate a normalized enrichment level. In someembodiments, a LE encoded in the test construct provides (or a cellgenetically modified and/or transduced with a retroviral particle (e.g.lentiviral particle) having a genome encoding the LE is capable ofproviding, is adapted for, possesses the property of, and/or is modifiedfor) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, orbetween 1.5 fold and 25-fold normalized enrichment level, or between3-fold and 20-fold normalized enrichment level, or between 5-fold and25-fold normalized enrichment level, or between 5-fold and 20-foldnormalized enrichment level, or between 5-fold and 15-fold normalizedenrichment level. Enrichment can be measured, for example, by directcell counting. Cutoff values can be based on a single test, or two,three, four, or five repeats, or based on many repeats. The cutoff canbe met when a lymphoproliferative element meets one or more repeattests, or meets or exceeds a cutoff for all repeats. In someembodiments, the enrichment is measured as log₂((normalized count dataon the test day+1)/(normalized count data on day 7+1)).

As illustrated in WO2019/055946, CLEs w ere identified from libraries ofconstructs that included constructs that encoded test chimericpolypeptides that were designed to comprise an intracellular domainbelieved to induce proliferation and/or survival of lymphoid or myeloidcells, and an anti-CD19 CAR that comprised an intracellular activatingdomain but not a co-stimulatory domain. Preactivation, which wasperformed overnight at 37° C., was performed in a preactivation reactionmixture comprising PBMCs, a commercial media for lymphocytes (CompleteOpTmizer™ CTS™ T-Cell Expansion SFM), recombinant human interleukin-2(100IU/ml) and anti-CD3 Ab (OKT3) (50 ng/ml). Following preactivation,transduction was performed overnight at 37° C. after addition of testand control lentiviral particles to the preactivation reaction mixturesat a multiplicity of infection (MOI) of 5. Some control lentiviralparticles contained constructs encoding polypeptides with extracellularand transmembrane domains but no intracellular domains. In contrast, thetest lentiviral particles contained constructs encoding polypeptideswith extracellular and transmembrane domains and either one or twointracellular domains. Following transduction. Complete OpTmizer™ CTS™T-Cell Expansion SFM was added to dilute the reaction mixture 5- to20-fold and the cells were cultured for up to 45 days at 37° C. Afterday 7 post-transduction, cultures were either “fed” additionaluntransduced donor matched PBMCs or not (“unfed”). No additionalcytokines (e.g. IL-2, IL-7, or IL-15 and no other lymphoid mitogenicagent) were added to these cultures that were not present in thecommercial media, after the transduction reaction mixtures wereinitially formed. Expansion was measured by analyzing enrichment of cellcounts actually counted as nucleic acid sequence counts of uniqueidentifiers for each construct in the mixed cultured PBMC cellpopulations, such that enrichment was positive as calculated as thelogarithm in base 2 of the ratio between normalized count at the lastday for analysis plus one to the count at day 7 plus one. Additionaldetails regarding the tests performed to identify the LEs areillustrated in WO2019/055946, including experimental conditions.

As illustrated in WO2019/055946, test constructs were identified as CLEsbecause the CLEs induced proliferation/expansion in these fed or unfedcultures without added cytokines such as IL-2 between days 7 and day 21,28, 35, and/or 42. For example, as illustrated in WO2019/055946,effective CLEs were identified by identifying test CLEs that providedincreased expansion of these in vitro cultures, whether fed or unfedwith untransduced PBMCs, between day 7 and day 21, 28, 35, and/or 42post-transduction, compared to control constructs that did not includeany intracellular domains. WO2019/055946 discloses that at least one andtypically more than one test CLE that included an intracellular domainfrom a test gene provided more expansion than every control constructthat was present at day 7 post-transduction, that did not include anintracellular domain. WO2019/055946 further provides a statisticalmethod that was used to identify exceptionally effective genes withrespect to a first intracellular domain, and one or more exemplaryintracellular domain(s) from these genes. The method used aMann-Whitney-Wilcoxon test and a false discovery cutoff rate of lessthan 0.1 or less than 0.05. WO2019/055946 identified especiallyeffective genes for the first intracellular domain or the secondintracellular domain, for example, by analyzing scores for genescalculated as combined score for all constructs with that gene. Suchanalysis can use a cutoff of greater than 1, or greater than negativecontrol constructs without any intracellular domains, or greater than 2,as shown for some of the tests disclosed in WO2019/055946.

In another embodiment, the LE provides, is capable of providing and/orpossesses the property of (or a cell genetically modified and/ortransduced with the LE is capable of providing, is adapted for,possesses the property of, and/or is modified for) driving T cellexpansion in vivo. For example, the in vivo test can utilize a mousemodel and measure T cell expansion at 15 to 25 days in vivo, or at 19 to21 days in vivo, or at approximately 21 days in vivo, after T cells arecontacted with lentiviral vectors encoding the LEs, are introduced intothe mice, as disclosed in WO2019/055946,

In exemplary aspects and embodiments that include a LE, which typicallyinclude a CAR, such as methods provided herein for geneticallymodifying, genetically modified and/or transduced cells, and usesthereof, the genetically modified cell is modified so as to possess newproperties not previously possessed by the cell before geneticmodification and/or transduction. Such a property can be provided bygenetic modification with a nucleic acid encoding a CAR or a LE, and inillustrative embodiments both a CAR and a LE. For example, in certainembodiments, the genetically modified and/or transduced cell is capableof, is adapted for, possesses the property of, and/or is modified forsurvival and/or proliferation in ex vivo culture for at least 7, 14, 21,28, 35, 42, or 60 days or from between day 7 and day 14, 21, 28, 35, 42or 60 post-transduction, in the absence of added IL-2 or in the absenceof added cytokines such as IL-2, IL-15, or IL-7, and in certainillustrative embodiments, in the presence of the antigen recognized bythe CAR where the method comprises genetically modifying using aretroviral particle having a pseudotyping element and optionally aseparate or fused activation domain on its surface and typically doesnot require pre-activation.

By capable of enhanced survival and/or proliferation in certainembodiments, it is meant that the genetically modified and/or transducedcell exhibits, is capable of, is adapted for, possesses the property of,and/or is modified for improved survival or expansion in ex vivo or invitro culture in culture media in the absence of one or more addedcytokines such as IL-2, IL-15, or IL-7, or added lymphocyte mitogenicagent, compared to a control cell(s) identical to the geneticallymodified and/or transduced cell(s) before it was genetically modifiedand/or transduced or to a control cell that was transduced with aretroviral particle identical to an on-test retroviral particle thatcomprises an LE or a putative LE, but without the LE or theintracellular domains of the LE, wherein said survival or proliferationof said control cell(s) is promoted by adding said one or morecytokines, such as IL-2, IL-15, or IL-7, or said lymphocyte mitogenicagent to tire culture media. By added cytokine or lymphocyte mitogenicagent, it is meant that cytokine or lymphocyte mitogenic agent is addedfrom an exogenous source to a culture media such that the concentrationof said cytokine or lymphocyte mitogenic agent is increased in theculture media during culturing of the cell(s) compared to the initialculture media, and in some embodiments can be absent from the initialculture media before said adding. By “added” or “exogenously added”, itis meant that such cytokine or lymphocyte mitogenic agent is added to alymphocyte media used to culture the genetically modified and/ortransduced cell after the genetically modifying, where the culture mediamay or may not already possess tire cytokine or lymphocyte mitogenicagent. All or a portion of the media that includes a mixture of multiplemedia components is typically stored and in illustrative embodiments hasbeen shipped to a site where the culturing takes place, without theexogenously added cytokine(s) or lymphocyte mitogenic agent(s). Thelymphocyte media in some embodiments is purchased from a supplier, and auser such as a technician not employed by the supplier and not locatedwithin a supplier facility, adds the exogenously added cytokine orlymphocyte mitogenic agent to the lymphocyte media and then thegenetically modified and/or transduced cells are cultured in thepresence or absence of such exogenously added cytokine or lymphocytemitogenic agent.

In some embodiments, improved or enhanced survival, expansion, and/orproliferation can be shown as an increase in the number of cellsdetermined by sequencing DNA from cells transduced with retroviralparticle (e.g. lentiviral particle) having a genome encoding CLEs andcounting the occurrences of sequences present in unique identifiers fromeach CLE. In some embodiments, improved survival and/or improvedexpansion can be determined by counting the cells directly, for examplewith a hemocytometer or a cell counter, at each time point. In someembodiments, improved survival and/or improved expansion and/orenrichment can be calculated by dividing the number of cells at thelater time point (day 21, 28, 35, and/or day 45) by the number of cellsat day 7 for each construct. In some embodiments, the cells can becounted by hemocytometer or cell counters. In some embodiments, theenrichment level determined using the nucleic acid counts or the cellcounts of each specific test construct can be normalized to theenrichment level of the respective control construct, i.e., theconstruct with the same extracellular domain and transmembrane domainbut lacking the intracellular domains present in the test construct. Inthese embodiments, the LE encoded in the construct provides (or a cellgenetically modified and/or transduced with a retroviral particle (e.g.lentiviral particle) having a genome encoding the LE is capable ofproviding, is adapted for, possesses the property of, and/or is modifiedfor) at least a 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or 10-fold normalized enrichment level, orbetween 1.5 fold and 25-fold normalized enrichment level, or between3-fold and 20-fold normalized enrichment level, or between 5-fold and25-fold normalized enrichment level, or between 5-fold and 20-foldnormalized enrichment level, or between 5-fold and 15-fold normalizedenrichment level.

In illustrative embodiments of any of the methods, uses, geneticallymodified T cells and/or NK cells, and other composition aspects providedherein that include a lymphoproliferative element, thelymphoproliferative element can include an intracellular domain or afragment thereof that includes an intracellular signaling domain fromany of the genes having a P3 signaling domain with or without a P4domain, or from any of the genes having a P4 domain wherein the P3domain was a linker, in the CLEs identified in Tables 4 to 8 herein,which promote T cell, e.g. CAR-T cell, expansion in vivo. Inillustrative embodiments of any of the methods, uses, and compositionaspects provided herein that include a lymphoproliferative elementhaving a P3 and P4 domain, the lymphoproliferative element can includeat the P4 position, an intracellular domain or a fragment thereof thatincludes a signaling domain from any of the genes having a P4 signalingdomain in constructs having a P3 and a P4 signaling domain in the CLEsidentified in Tables 4 to 8 herein, which promote T cell, e.g. CAR-Tcell, expansion in vivo. In illustrative embodiments of any of themethods, uses, and composition aspects provided herein that include alymphoproliferative element, the lymphoproliferative element can includean intracellular domain or a fragment thereof that includes a signalingdomain from any of the genes having a P3 signaling domain and asignaling domain from any of the genes having a P4 domain in the sameCLE, in illustrative embodiments in the P3 and P4 positionsrespectively, in any of the CLEs identified in Tables 4 to 8 herein,which promote T cell, e.g. CAR-T cell, expansion in vivo. In any of theCLEs of embodiments provided in this paragraph, the P2 domain can befrom any of the genes identified as having a P2 part in CLEs found inTables 4 to 8 herein. Furthermore, the CLEs can include in someillustrative embodiments a P1 domain from Tables 4 to 8.

In illustrative embodiments of any of the methods, uses, geneticallymodified T cells and/or NK cells, and other composition aspects providedherein that include a lymphoproliferative element, thelymphoproliferative element can include a P3 signaling domain from anyof the CLEs identified in Tables 4 to 8 herein, which promote T cell,e.g. CAR-T cell, expansion in vivo, or a P4 signaling domain in aconstruct having no P3 signaling domain, from any of the CLEs identifiedin Tables 4 to 8 herein, which promote T cell, e.g. CAR-T cell,expansion in vivo. In illustrative embodiments of any of the methods,uses, and composition aspects provided herein that include alymphoproliferative element having a P3 and P4 domain, thelymphoproliferative element can include at the P4 position, a P4signaling domain in constructs having a P3 and a P4 signaling domain inthe CLEs identified in Tables 4 to 8 herein, which promote T cell, e.g.CAR-T cell, expansion in vivo. In illustrative embodiments of any of themethods, uses, and composition aspects provided herein that include alymphoproliferative element, the lymphoproliferative element can includea P3 signaling domain and a P4 signaling domain in the P3 and P4positions respectively, from any one of the CLEs identified in Tables 4to 8 herein, which promote T cell, e.g. CAR-T cell, expansion in vivo.Furthermore, the CLEs can include in some illustrative embodiments, a P1domain from Tables 4 to 8. In any of the CLEs of embodiments provided inthis paragraph, the P2 domain can comprise or be any P2 domain from aCLE found in Tables 4 to 8 herein, or in illustrative embodiments, alymphoproliferative element can include a P2 domain. P3 domain and P4domain, and optionally P1 domain, all from the same CLE identified inTables 4 to 8 herein. In certain illustrative embodiments of any of themethods, uses, genetically modified T cells and/or NK cells, and othercomposition aspects provided herein that include a lymphoproliferativeelement, the lymphoproliferative element can have P3 and P4 domainsS121-S212 or S186-S053, or P2, P3, and P4 domains T001-S121-S212 orT044-S186-S053 optionally with a P1 domain E008 or E006.

In some embodiments, the lymphoproliferative element can include acytokine receptor or a fragment that includes a signaling domainthereof. In some embodiments, the cytokine receptor can be CD27, CD40,CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2,IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL2RA, IL2RB, IL2RG, IL3RA,IL4R, IL5RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA,IL12RB1, IL13R, IL13RA1, IL13RA2, IL15R, IL15RA, IL17RA, IL17RB, IL17RC,IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27R,IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TGFβR, TGFβ decoy receptor,TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18. In some embodiments,the cytokine receptor can be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R,EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP,IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST,IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL15RA, IL17RA,IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1,IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9,TNFRSF14, or TNFRSF18.

In illustrative embodiments, the lymphoproliferative element cancomprise an intracellular domain from the cytokine receptors CD27, CD40,CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR2, IL1R1, IL1RL1,IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1,IL13RA1, IL13RA2, IL15RA, IL17RB, IL18R1, IL18RAP, IL20RB, IL22RA1,IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9,TNFRSF14, or TNFRSF18In illustrative embodiments, the intracellulardomain in a lymphoproliferative element comprises a domain from CD40,CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R,IL10RB, IL11RA, IL12RB1, IL13RA2, IL18RAP, IL31RA, MPL, MYD88, TNFRSF14,or TNFRSF18, which were present in constructs that showed particularlynoteworthy enrichments in an initial screen and a repeated screen asdisclosed in WO2019/055946.

In illustrative embodiments, the lymphoproliferative element cancomprise a costimulatory domain from CD27, CD28, OX40 (also referred toas TNFRSF4), GITR (also referred to as TNFRSF18), or HVEM (also referredto as TNFRSF14). In some embodiments, a lymphoproliferative elementcomprising a costimulatory domain from OX40 does not comprise anintracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30,GITR, or HVEM. In some embodiments, a lymphoproliferative elementcomprising a costimulatory domain from GITR docs not comprise anintracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, orHVEM. In some embodiments, a lymphoproliferative element comprising acostimulatory domain from CD28 does not comprise an intracellular domainfrom CD3Z, 4-1BB, ICOS, CD27, BTLA, CD30, or HVEM. In some embodiments,a lymphoproliferative element comprising a costimulatory domain fromOX40, CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR, or HVEM does notcomprise a coiled-coil spacer domain N-terminal of the transmembranedomain. In some embodiments, a lymphoproliferative element comprising acostimulatory domain from GITR does not comprise an intracellular domainfrom CD3Z that is N-terminal of the costimulatory domain of GITR.

In certain illustrative embodiments, the lymphoproliferative elementcomprises an intracellular domain of CD40, MPL and IL2Rb. In someembodiments, the lymphoproliferative element can be other than acytokine receptor. In some embodiments, the lymphoproliferative elementother than a cytokine receptor can include an intracellular signalingdomain from CD2, CD3D, CD3G, CD3Z, CD4, CD8RA, CD8RB, CD28, CD79A,CD79B, FCER1G, FCGR2A, FCGR2C, or ICOS

In some embodiments, a lymphoproliferative element, including a CLE,comprises an intracellular activating domain as disclosed hereinabove.In some illustrative embodiments a lymphoproliferative element is a CLEcomprising an intracellular activating domain comprising anITAM-containing domain, as such, the CLE can comprise an intracellularactivating domain having at least 80%, 90%, 95%, 98%, or 100% sequenceidentity to the CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCERlG,FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domains provided herein wherein theCLE does not comprise an ASTR. In certain illustrative embodiments, theintracellular activating domain is an ITAM-containing domain from CD3D,CD3G, CD3Z, CD79A, CD79B, FCER1G, FCGR2A, or FCGR2C. CLEs comprisingthese intracellular activating domains are illustrated in WO2019/055946,as being effective at promoting proliferation of PBMCs ex vivo incultures in the absence of exogenous cytokines such as exogenous IL-2.In some embodiments, provided herein are CLEs comprising anintracellular domain from CD3D, CD3G, CD3Z, CD79A, FCER1G.

In some embodiments, one or more domains of a lymphoproliferativeelement is fused to a modulatory domain, such as a co-stimulatorydomain, and/or an intracellular activating domain of a CAR. In someembodiments of the composition and method aspects for transducinglymphocytes in whole blood, one or more intracellular domains of alymphoproliferative element can be part of the same polypeptide as a CARor can be fused and optionally functionally connected to some componentsof CARs. In still other embodiments, an engineered signaling polypeptidecan include an ASTR, an intracellular activation domain (such as a CD3zeta signaling domain), a co-stimulatory domain, and alymphoproliferative domain. Further details regarding co-stimulatorydomains, intracellular activating domains, ASTRs and other CAR domains,are disclosed elsewhere herein.

In some embodiments, the lymphoproliferative element is not apolypeptide, but rather comprises an inhibitory RNA. In someembodiments, methods, uses, compositions, and products of processesaccording to any aspect herein include both a lymphoproliferativeelement comprising an inhibitory RNA and a lymphoproliferative elementthat is an engineered signaling polypeptide. In embodiments where alymphoproliferative element is or includes an inhibitory RNA, ormultiple inhibitory RNAs, the inhibitory RNA or multiple inhibitoryRNAs, can have any of the structures identified elsewhere herein, forexample in the Inhibitory RNA Molecules section herein. In someembodiments, the inhibitory RNA can be a miRNA that stimulates the STAT5pathway typically by potentiating activation of STAT5 by degrading orcausing down-regulation of a negative regulator in the SOCS pathway.Inhibitory RNA lymphoproliferative elements can target any of the mRNAsidentified in the Inhibitory RNA Molecules section herein or elsewhereherein.

In illustrative embodiments, as exemplified herein, such inhibitory RNA(e.g. miRNAs) can be located in introns in packaging cells and/or areplication incompetent recombinant retroviral particle genome and/or aretroviral vector, typically with expression driven by a promoter thatis active in a T cell and/or NK cell. Not to be limited by theory,inclusion of introns in transcription units are believed to result inhigher expression and/or stability of transcripts. As such, the abilityto place miRNAs within introns of a retroviral genome adds to theteachings of the present disclosure that overcome challenges in theprior art of trying to get maximum activities into the size restrictionsof a retroviral, such as a lentivirus genome. In some embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 miRNAs, in illustrative embodiments between 2and 5, for example 4 miRNAs, one or more of which each bind nucleicacids encoding one or more of the targets disclosed herein, can beincluded in the recombinant retroviral genome and delivered to a targetcell, for example T cells and/or NK cells, using methods providedherein. In fact, as provided herein 1, 2, 3, or 4 miRNAs can bedelivered in a single intron such as the EF1-a intron.

In some embodiments, the lymphoproliferative element comprises MPL, oris MPL, or a variant and/or fragment thereof, including a variant and/orfragment that includes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or100% of the intracellular domain of MPL, with or without a transmembraneand/or extracellular domain of MPL, and/or has at least 75, 80, 85, 90,95, 96, 97, 98, 99, or 100% sequence identity to the intracellulardomain of MPL, with or without a transmembrane and/or extracellulardomain of MPL, wherein the variant and/or fragment retains the abilityto promote cell proliferation of PBMCs, and in some embodiments T cells.In illustrative embodiments, the lymphoproliferative element comprisesan intracellular domain of MPL, or a variant or fragment thereof thatincludes at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of theintracellular domain of MPL, and the lymphoproliferative element doesnot comprise a transmembrane domain of MPL. In some embodiments, thelymphoproliferative element comprises an intracellular domain of MPL, ora variant or fragment thereof that includes at least 75, 80, 85, 90, 95,96, 97, 98, 99, or 100% of tire intracellular domain of MPL, and thelymphoproliferative element comprises a transmembrane domain of MPL. Insome embodiments, a cell expressing the lymphoproliferative elementcomprising an intracellular and transmembrane domain of MPL can becontacted with, exposed to, or treated with eltrombopag. Not to belimited by theory, eltrombopag binds to the transmembrane domain of MPLand induces the activation of the intracellular domain of MPL. In someembodiments, an MPL fragment included in the compositions and methodsherein has and/or retains a JAK-2 binding domain. In some embodiments,an MPL fragment included herein has or retains the ability to activate aSTAT. The full intracellular domain of MPL is SEQ ID NO:283 (part S186as illustrated in WO2019/055946). MPL is the receptor forthrombopoietin. Several cytokines such as thrombopoietin and EPO arereferred to m the literature and herein as either a hormone or acytokine.

In some embodiments, which provide separate aspects of the presentdisclosure, provided herein are chimeric polypeptides that are chimericlymphoproliferative elements (CLEs), as well as isolated polynucleotidesand nucleic acid sequences that encode the same. CLEs can include any ofthe domains and/or domains derived from specific genes discussed in thesection. Similarly, the isolated polynucleotides and nucleic acidsequences encoding CLEs can encode as part of the CLE any of the domainsand/or domains derived from specific genes discussed in this section.

Lymphoproliferative elements provided herein typically include atransmembrane domain. For example, the transmembrane domain can have80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any oneof the transmembrane domains from the following genes and representativesequences disclosed in WO2019/055946: CD8 beta, CD4, CD3 zeta, CD28,CD134, CD7, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A CD8B, CD27, CD28,CD40, CD79A, CD79B, CRLF2, CRLF2, CSF2RA, CSF2RB, CSF2RB, CSF3R, EPOR,FCER1G, FCGR2C, FCGRA2, GHR, GHR, ICOS, IFNAR, IFNAR2, IFNGR1, IFNGR2,IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R,IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD,IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA,IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9,TNFRSF14, and TNFRSF18. Transmembrane (TM) domains suitable for use inany engineered signaling polypeptide include, but are not limited to,constitutively active cytokine receptors, the TM domain from LMP1, andTM domains from type 1 TM proteins comprising a dimerizing motif, asdiscussed in more detail herein. In any of the aspects disclosed hereincontaining the transmembrane domain from a type I transmembrane protein,the transmembrane domain can be a Type I growth factor receptor, ahormone receptor, a T cell receptor, or a TNF-family receptor.

Eltrombopag is a small molecule activator of the thrombopoietin receptorMPL (also known as TPOR). In some aspects a cell expressing an LEcomprising a MPL transmembrane domain, can be exposed to or contactedwith eltrombopag, or a patient or subject to which such a cell has beeninfused, can be treated with eltrombopag. Upon said contacting ortreating, the proliferative and/or survival properties of the LE areactivated and provided to the cell, thereby increasing survival and/orproliferation of the cell compared to the absence of the eltrombopag.Not to be limited by theory, binding of eltrombopag occurs in thetransmembrane domain and can activate one or more intracellular domainsthat are part of the same polypeptide. A skilled artisan will understandthe amount of eltrombopag to be used to activate a CLE comprising a MPLtransmembrane domain.

In some embodiments, CLEs include both an extracellular portion and atransmembrane portion that is from the same protein, in illustrativeembodiments the same receptor, either of which in illustrativeembodiments is a mutant, thus forming an extracellular and transmembranedomain. These domains can be from a cytokine receptor, or a mutantthereof, or a hormone receptor, or a mutant thereof in some embodimentsthat have been reported to be constitutively active when expressed atleast in some cell types. In illustrative embodiments, suchextracellular and transmembrane domains do not include a ligand bindingregion. It is believed that such domains do not bind a ligand whenpresent in CLEs and expressed in B cells, T cells, and/or NK cells.Mutations in such receptor mutants can occur in the transmembrane regionor in the extracellular juxtamembrane region. Not to be limited bytheory, a mutation in at least some extracellular—transmembrane domainsof CLEs provided herein, are responsible for signaling of the CLE in theabsence of ligand, by bringing activating chains together that are notnormally together, or by changing the confirmation of a linkedtransmembrane and/or intracellular domain.

Exemplary extracellular and transmembrane domains for CLEs ofembodiments that include such domains, in illustrative embodiments, areextracellular regions, typically less than 30 amino acids of themembrane-proximal extracellular domains along with transmembrane domainsfrom mutant receptors that have been reported to be constitutive, thatis not require ligand binding for activation of an associatedintracellular domain. In illustrative embodiments, such extracellularand transmembrane domains include IL7RA Ins PPCL, CRLF2 F232C, CSF2RBV449E, CSF3R T640N, EPOR L251C I252C, GHR E260C I270C, IL27RA F523C, andMPL S505N. In some embodiments, the extracellular and transmembranedomain does not comprise more than 10, 20, 25 30 or 50 consecutive aminoacids that are identical in sequence to a portion of the extracellularand/or transmembrane domain of IL7RA, or a mutant thereof. In someembodiments, the extracellular and transmembrane domain is other thanIL7RA Ins PPCL. In some embodiments, the extracellular and transmembranedoes not comprise more than 10, 20, 25, 30, or 50 consecutive aminoacids that are identical in sequence to a portion of the extracellularand/or transmembrane domain of IL15R.

In one embodiment of this aspect, an LE provided herein comprises anextracellular domain, and in illustrative embodiments, the extracellulardomain comprises a dimerizing motif. In illustrative embodiments of thisaspect, the extracellular domain comprises a leucine zipper. In someembodiments, the leucine zipper is from a jun polypeptide, for examplec-jun. In certain embodiments the c-jun polypeptide is the c-junpolypeptide region of ECD-11.

In embodiments of any of these aspects and embodiments wherein thetransmembrane domain is a type I transmembrane protein, thetransmembrane domain can be a Type I growth factor receptor, a hormonereceptor, a T cell receptor, or a TNF-family receptor. In an embodimentof any of the aspects and embodiments wherein the chimeric polypeptidecomprises an extracellular domain and wherein the extracellular domaincomprises a dimerizing motif, the transmembrane domain can be a Type Icytokine receptor, a hormone receptor, a T cell receptor, or aTNF-family receptor.

Exemplary transmembrane domains include any transmembrane domain thatwas illustrated in WO2019/055946. In some embodiments, the transmembranedomain is from CD4, CD8RB, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C,GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL2RG, IL3RA, IL5RA,IL6ST, IL7RA, IL10RB, IL11RA, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RE,IL18R1, IL18RAP, IL20RA, IL22RA1, IL31RA, LEPR, PRLR, and TNFRSF8, ormutants thereof that are known to promote signaling activity in certaincell types if such mutants are present in the constructs provided inWO2019/055946. In some embodiments, the transmembrane domain is fromCD40, ICOS, FCGR2C, PRLR, IL3RA, or IL6ST.

In some embodiments, the extracellular and transmembrane domain is theviral protein LMP1, or a mutant and/or fragment thereof. LMP1 is amultispan transmembrane protein that is known to activate cell signalingindependent of ligand when targeted to lipid rafts or when fused to CD40(Kaykas et al. EMBO J. 20: 2641 (2001)). A fragment of LMP1 is typicallylong enough to span a plasma membrane and to activate a linkedintracellular domain(s). For example, the LMP1 can be between 15 and386, 15 and 200, 15 and 150, 15 and 100, 18 and 50, 18 and 30, 20 and200, 20 and 150, 20 and 50, 20 and 30, 20 and 100, 20 and 40, or 20 and25 amino acids. A mutant and/or fragment of LMP1 when included in a CLEprovided herein, retains its ability to activate an intracellulardomain. Furthermore, if present, the extracellular domain includes atleast 1, but typically at least 4 amino acids and is typically linked toanother functional polypeptide, such as a clearance domain, for example,an eTag. In some embodiments, the lymphoproliferative element comprisesan LMP1 transmembrane domain. In illustrative embodiments, thelymphoproliferative element comprises an LMP1 transmembrane domain andthe one or more intracellular domains do not comprise an intracellulardomain from TNFRSF proteins (i.e. CD40, 4-IBB, RANK, TACI, OX40, CD27,GITR, LTR, and BAFFR), TLR1 to TLR13, integrins, FcγRIII, Dectin1,Dectin2, NOD1, NOD2, CD16, IL-2R, Type I II interferon receptor,chemokine receptors such as CCR5 and CCR7, G-protein coupled receptors,TREM1, CD79A, CD79B, Ig-alpha, IPS-1, MyD88, RIG-1, MDA5, CD3Z,MyD88ΔTIR, TRIF, TRAM, TIRAP, MAL, BTK, RTK, RAC1, SYK, NALP3 (NLRP3),NALP3ΔLRR, NALP1, CARD9, DAI, IPAG, STING, Zap70, or LAT.

In other embodiments of CLEs provided herein, the extracellular domainincludes a dimerizing moiety. Many different dimerizing moietiesdisclosed herein can be used for these embodiments. In illustrativeembodiments, the dimerizing moieties are capable of homodimerizing. Notto be limited by theory, dimerizing moieties can provide an activatingfunction on intracellular domains connected thereto via transmembranedomains. Such activation can be provided, for example, upon dimerizationof a dimerizing moiety, which can cause a change in orientation ofintracellular domains connected thereto via a transmembrane domain, orwhich can cause intracellular domains to come into proximity. Anextracellular domain with a dimerizing moiety can also serve a functionof connecting a recognition tag to a cell expressing a CLE. In someembodiments, the dimerizing agent can be located intracellularly ratherthan extracellularly. In some embodiments, more titan one or multiplesof dimerizing domains can be used.

Extracellular domains for embodiments where extracellular domains have adimerizing motif, are long enough to form dimers, such as leucine zipperdimers. As such, extracellular domains that include a dimerizing moietycan be from 15 to 100, 20 to 50, 30 to 45, or 35 to 40 amino acids, ofin illustrative embodiments is a c-Jun portion of a c-Jun extracellulardomain. Extracellular domains of polypeptides that include a dimerizingmoiety, may not retain other functionalities. For example, for leucinezippers embodiments, such leucine zippers are capable of forming dimersbecause they retain a motif of leucines spaced 7 residues apart along analpha helix. However, leucine zipper moieties of certain embodiments ofCLEs provided herein, may or may not retain their DNA binding function.

A spacer of between 1 and 4 alanine residues can be included in CLEsbetween the extracellular domain that has a dimerizing moiety, and thetransmembrane domain. Not to be limited by theory, it is believed thatthe alanine spacer affects signaling of intracellular domains connectedto the leucine zipper extracellular region via the transmembrane domain,by changing the orientation of tire intracellular domains.

The first and optional second intracellular domains of CLEs providedherein, are intracellular signaling domains of genes that are known inat least some cell types, to promote proliferation, survival(anti-apoptotic), and/or provide a co-stimulatory signal that enhancesproliferative potential or resistance to cell death. As such, theseintracellular domains can be intracellular domains fromlymphoproliferative elements and co-stimulatory domains provided herein.Some of tire intracellular domains of candidate chimeric polypeptidesare known to activate JAK1/JAK2, JAK3, STAT1, STAT2, STAT3, STAT4,STAT5, and STAT6 signaling. Conserved motifs that are found inintracellular domains of cytokine receptors that are responsible forthis signaling are known (see e.g., Morris et al., “The moleculardetails of cytokine signaling via the JAK/STAT pathway,” Protein Science(2018) 27:1984-2009). The Box1 and Box2 motifs are involved in bindingto JAKs and signal transduction, although the Box2 motif presence is notalways required for a proliferative signal (Murakami et al. Proc NatlAcad Sci USA. 1991 Dec. 15; 88(24):11349-53; Fukunaga et al. EMBO J.1991 October; 10(10):2855-65; and O'Neal and Lee. Lymphokine CytokineRes. 1993 October; 12(5):309-12). Accordingly, in some embodiments alymphoproliferative element herein is a transgenic BOX 1-containingcytokine receptor that includes an intracellular domain of a cytokinereceptor comprising a Box1 Janus kinase (JAK)-binding motif, optionallya Box2 JAK-binding motif, and a Signal Transducer and Activator ofTranscription (STAT) binding motif comprising a tyrosine residue. Manycytokine receptors have hydrophobic residues at positions −1, −2, and −6relative to the Box1 motif, that form a “switch motif,” which isrequired for cytokine-induced JAK2 activation but not for JAK2 binding(Constantinescu et al. Mol Cell. 2001 February; 7(2):377-85; and Huanget al. Mol Cell. 2001 December; 8(6):1327-38). Accordingly, in certainembodiments of the transgenic BOX 1-containing cytokine receptorlymphoproliferative element has a switch motif, which in illustrativeembodiments has one or more, and preferably all hydrophobic residues atpositions −1, −2, and −6 relative to the Box1 motif. In certainembodiments, the Box1 motif an ICD of a lymphoproliferative element islocated proximal to the transmembrane (TM) domain (for example between 5and 15 or about 10 residues downstream from the TM domain) relative tothe Box2 motif, which is located proximal to the transmembrane domain(for example between 10 and 50 residues downstream from the TM domain)relative to the STAT binding motif. The STAT binding motif typicallycomprising a tyrosine residue, the phosphorylation of which affectsbinding of a STAT to the STAT binding motif of the lymphoproliferativeelement. In some embodiments, the ICDs comprising multiple STAT bindingmotifs where multiple STAT binding motifs are present in a native ICD(e.g. EPO receptor and IL-6 receptor signaling chain (gp130).

Intracellular domains from IFNAR1, IFNGR1, IFNLR1, IL2RB, IL4R, IL5RB,IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL21R, IL27R, IL31RA, LIFR, and OSMRare known in the art to activate JAK1 signaling. Intracellular domainsfrom CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNGR2, IL3RA, IL5RA,IL6ST, IL20RA, IL20RB, IL23R, IL27R, LEPR, MPL, and PRLR are known inthe art to activate JAK2. Intracellular domains from IL2RG are known inthe art to activate JAK3. Intracellular domains from GHR, IFNAR1,IFNAR2, IFNGR1, IFNGR2, IL2RB, IL2RG, IL4R, IL5RA, IL5RB, IL7RA, IL9R,IL21R, IL22RA1, IL31RA, LIFR, MPL, and OSMR are known in the art toactivate STAT1. Intracellular domains from IFNAR1 and IFNAR2 are knownin the art to activate STAT2. Intracellular domains from GHR, IL2RB,IL2RG, IL6R, IL7RA, IL9R, IL10RA, IL10RB, IL21R, IL22RA1, IL23R, IL27R,IL31RA, LEPR, LIFR, MPL, and OSMR are known in the art to activateSTAT3. Intracellular domains from IL12RB1 are known in the art toactivate STAT4. Intracellular domains from CSF2RA, CSF2RB, CSF3R, EPOR,GHR, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL15RA,IL20RA, IL20RB, IL21R, IL22RA1, IL31RA, LIFR, MPL, OSMR, and PRLR areknown in the art to activate STAT5. Intracellular domains from IL4R andOSMR are known in the art to activate STAT6. The genes and intracellulardomains thereof that are found in a first intracellular domain are thesame as the optional second intracellular domain, except that if thefirst and second intracellular domain are identical, then at least one,and typically both the transmembrane domain and the extracellular domainare not from the same gene.

In some embodiments, all domains of a CLE are other than an IL-7receptor, or a mutant thereof, and/or a fragment thereof that has atleast 10, 15, 20, or 25 contiguous amino acids of IL-7 receptor, orother than an IL-15 receptor, or a mutant thereof, and/or a fragmentthereof that has at least 10, 15, 20, or 25 contiguous amino acids ofIL-15 receptor. In some embodiments, a CLE docs not comprise acombination of first intracellular domain and second intracellulardomain of CD40 and MyD88.

In illustrative embodiments, CLEs include a recognition and/orelimination domain. Details regarding recognition and/or eliminationdomains are provided in other sections herein. Any of the recognitionand/or elimination domains provided herein can be part of a CLE.Typically the recognition domain is linked to the N terminus of theextracellular domain. Not to be limited by theory, in some embodiments,the extracellular domain includes the function of providing a linker, inillustrative embodiments a flexible linker, linking a recognition domainto a cell that expresses the CLE.

Furthermore, polynucleotides that include a nucleic acid sequenceencoding a CLE provided herein, also typically comprise a signalsequence to direct expression to the plasma membrane. Exemplary signalsequences are provided herein in other sections. Elements can beprovided on the transcript such that both a CAR and CLE are expressedfrom the same transcript in certain embodiments.

In any aspects or embodiments wherein the extracellular domain of a CLEcomprises a dimerizing motif, the dimerizing motif can be selected fromthe group consisting of: a leucine zipper motif-containing polypeptide,CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, aswell as mutants and/or active fragments thereof that retain the abilityto dimerize. In any of the aspects and embodiments herein wherein theextracellular domain of a CLE comprises a dimerizing motif, thedimerizing motif can require a dimerizing agent, and the dimerizingmotif and associated dimerizing agent can be selected from the groupconsisting of: FKBP and rapamycin or analogs thereof, GyrB andcoumermycin or analogs thereof, DHFR and methotrexate or analogsthereof, or DmrB and AP20187 or analogs thereof, as well as mutantsand/or active fragments of the recited dimerizing proteins that retainthe ability to dimerize. In some aspects and illustrative embodiments, alymphoproliferative element is constitutively active, and is other thana lymphoproliferative element that requires a dimerizing agent foractivation.

In illustrative embodiments of any aspects or embodiments herein whereinthe extracellular domain of a CLE comprises a dimerizing motif, theextracellular domain can comprise a leucine zipper motif. In someembodiments, tire leucine zipper motif is from a jun polypeptide, forexample c-jun. In certain embodiments the c-jun polypeptide is the c-junpolypeptide region of ECD-11.

Internally dimerizing and/or multimerizing lymphoproliferative elementsin one embodiment are an integral part of a system that uses a dimericanalog of the lipid permeable immunosuppressant drug, FK506, which losesits normal bioactivity while gaining the ability to crosslink moleculesgenetically fused to the FK506-binding protein, FKBP12. By fusing one ormore FKBPs and a myristoylation sequence to the cytoplasmic signalingdomain of a target receptor, one can stimulate signaling in a dimerizerdrug-dependent, but ligand and ectodomain-independent manner. Thisprovides the system with temporal control, reversibility using monomericdrug analogs, and enhanced specificity. The high affinity ofthird-generation AP20187/AP1903 dimerizer drugs for their bindingdomain, FKBP12 permits specific activation of tire recombinant receptorin vivo without the induction of non-specific side effects throughendogenous FKBP12. FKBP12 variants having amino acid substitutions anddeletions, such as FKBP12V36, that bind to a dimerizer drug, may also beused. In addition, the synthetic ligands are resistant to proteasedegradation, making them more efficient at activating receptors in vivothan most delivered protein agents.

Pseudotyping Elements

Many of the methods and compositions provided herein includepseudotyping elements. The pseudotyping of replication incompetentrecombinant retroviral particles with heterologous envelopeglycoproteins typically alters the tropism of a virus and facilitatesthe transduction of host cells. A pseudotyping element as used hereincan include a “binding polypeptide” that includes one or morepolypeptides, typically glycoproteins, that identify and bind the targethost cell, and one or more “fusogenic polypeptides” that mediate fusionof the retroviral and target host cell membranes, thereby allowing aretroviral genome to enter the target host cell. In some embodimentsprovided herein, pseudotyping elements are provided aspolypeptide(s)/protein(s), or as nucleic acid sequences encoding thepolypeptide(s)/protein(s).

In some embodiments, the pseudotyping element is the feline endogenousvirus (RD114) envelope protein, an oncoretroviral amphotropic envelopeprotein, an oncoretroviral ecotropic envelope protein, the vesicularstomatitis virus envelope protein (VSV-G) (SEQ ID NO: 336), the baboonretroviral envelope glycoprotein (BaEV) (SEQ ID NO: 337), the murineleukemia envelope protein (MuLV) (SEQ ID NO: 338), the influenzaglycoprotein HA surface glycoprotein (HA), the influenza glycoproteinneuraminidase (NA), the paramyxovirus Measles envelope protein H, theparamyxovirus Measles envelope protein F, and/or functional variants orfragments of any of these envelope proteins.

In some embodiments, the pseudotyping element can be wild-type BaEV. Notto be limited by theory, BaEV contains an R peptide that has been shownto inhibit transduction. In some embodiments, the BaEV can contain adeletion of the R peptide. In some embodiments, the BaEV can contain adeletion of the inhibitory R peptide after the nucleotides encoding theamino acid sequence HA, referred to herein as BaEVΔR (HA) (SEQ ID NO:339). In some embodiments, the BaEV can contain a deletion of theinhibitory R peptide after the nucleotides encoding the amino acidsequence HAM, referred to herein as BaEVΔR (HAM) (SEQ ID NO: 340).

In some embodiments, the pseudotyping element can be wild-type MuLV. Insome embodiments, the MuLV can contain one or more mutations to removethe furin-mediated cleavage site located between the transmembrane (TM)and surface (SU) subunits of the envelope glycoprotein. In someembodiments the MuLV contains the SUx mutation (MuLVSUx) (SEQ ID NO:453) which inhibits furin-mediated cleavage of MuLV envelope protein inpackaging cells. In certain embodiments the C-terminus of thecytoplasmic tail of the MuLV or MuLVSUx protein is truncated by 4 to 31amino acids. In certain embodiments the C-terminus of the cytoplasmictail of the MuLV or MuLVSUx protein is truncated by 4, 8, 12, 16, 20,24, 28, or 31 amino acids.

In some embodiments, the pseudotyping elements include a bindingpolypeptide and a fusogenic polypeptide derived from different proteins.For example, the replication incompetent recombinant retroviralparticles of the methods and compositions disclosed herein can bepseudotyped with the fusion (F) and/or hemagglutinin (H) polypeptides ofthe measles virus (MV), as non-limiting examples, clinical wildtypestrains of MV, and vaccine strains including the Edmonston strain(MV-Edm) (GenBank; AF266288.2) or fragments thereof. Not to be limitedby theory, both hemagglutinin (H) and fusion (F) polypeptides arebelieved to play a role in entry into host cells wherein the H proteinbinds MV to receptors CD46, SLAM, and Nectin-4 on target cells and Fmediates fusion of the retroviral and host cell membranes. In anillustrative embodiment, especially where the target cell is a T celland/or NK cell, the binding polypeptide is a Measles Virus H polypeptideand the fusogenic polypeptide is a Measles Virus F polypeptide.

In some studies, lentiviral particles pseudotyped with truncated F and Hpolypeptides had a significant increase in titers and transductionefficiency (Funke et al. 2008. Molecular Therapy. 16(8):1427-1436),(Frecha et al. 2008. Blood. 112(13):4843-4852). The highest titers wereobtained when the F cytoplasmic tail was truncated by 30 residues(referred to as MV(Ed)-FΔ30 (SEQ ID NO:313)). For the H variants,optimal truncation occurred when 18 or 19 residues were deleted(MV(Ed)-HΔ18 (SEQ ID NO:314) or MV(Ed)-HΔ19), although variants with atruncation of 24 residues with and without replacement of deletedresidues with alanine (MV(Ed)-HΔ24 (SEQ ID NO:315) and MV(Ed)-HΔ24+A)also resulted in optimal titers. Accordingly, in some embodiments,including those directed to transducing T cells and/or NK cells, thereplication incompetent recombinant retroviral particles of the methodsand compositions disclosed herein are pseudotyped with mutated orvariant versions of the measles virus fusion (F) and hemagglutinin (H)polypeptides, in illustrative examples, cytoplasmic domain deletionvariants of measles virus F and H polypeptides. In some embodiments, themutated F and H polypeptides are “truncated H” or “truncated F”polypeptides, whose cytoplasmic portion has been truncated, i.e. aminoacid residues (or coding nucleic acids of the corresponding nucleic acidmolecule encoding the protein) have been deleted. “HΔY” and “FΔX”designate such truncated H and F polypeptide, respectively, wherein “Y”refers to 1-34 residues that have been deleted from the amino terminiand “X” refers to 1-35 residues that have been deleted from the carboxytermini of the cytoplasmic domains. In a further embodiment, the“truncated F polypeptide” is FΔ24 or FΔ30 and/or the “truncated Hprotein” is selected from the group consisting of HΔ14, HΔ15, HΔ16,HΔ17, HΔ18, HΔ19, HΔ20, HΔ21+A, HΔ24 and HΔ24+4A, more preferably HΔ18or HΔ24. In an illustrative embodiment, the truncated F polypeptide isMV(Ed)-FΔ30 and the truncated H polypeptide is MV(Ed)-HΔ18.

In some embodiments, the pseudotyping element includes polypeptidesderived from different proteins. For example, the pseudotyping elementcan comprise an influenza protein hemagglutinin HA and/or aneuraminidase (NA). In certain embodiments the HA is from influenza Avirus subtype H1N1. In illustrative embodiments the HA is from H1N1 PR81934 in which the monobasic trypsin-dependent cleavage site has beenmutated to a more promiscuous multibasic sequence (SEQ ID NO:311). Incertain embodiments the NA is from influenza A virus subtype H10N7. Inillustrative embodiments the NA is from H10N7-HKWF446C-07 (SEQ IDNO:312).

In some embodiments, the viral particles are copseudotyped with envelopeglycoproteins from 2 or more heterologous viruses. In some embodiments,the viral particles are copseudotyped with VSV-G, or a functionalvariant or fragment thereof, and an envelope protein from RD114, BaEV,MuLV, influenza virus, measles virus, and/or a functional variant orfragment thereof. In some embodiments, the viral particles arecopseudotyped with VSV-G and the MV(Ed)-H glycoprotein or the MV(Ed)-Hglycoprotein with a truncated cytoplasmic domain. In illustrativeembodiments, the viral particles are copseudotyped with VSV-G andMV(Ed)-HΔ24. In certain embodiments, VSV-G is copseudotyped with MuLV orMuLV with a truncated cytoplasmic domain. In other embodiments, VSV-G iscopseudotyped with MuLVSUx or MuLVSUx with a truncated cytoplasmicdomain. In further illustrative embodiments, VSV-G is copseudotyped witha fusion of an antiCD3scFv to MuLV.

In some embodiments, the fusogenic polypeptide includes multipleelements expressed as one polypeptide. In some embodiments, the bindingpolypeptide and fusogenic polypeptide are translated from the sametranscript but from separate ribosome binding sites; in otherembodiments, the binding polypeptide and fusogenic polypeptide areseparated by a cleavage peptide site, which not to be bound by theory,is cleaved after translation, as is common in the literature, or aribosomal skip sequence. In some embodiments, the translation of thebinding polypeptide and fusogenic polypeptide from separate ribosomebinding sites results in a higher amount of the fusogenic polypeptide ascompared to the binding polypeptide. In some embodiments, the ratio ofthe fusogenic polypeptide to the binding polypeptide is at least 2; 1,at least 3:1, at least 4; 1, at least 5; 1, at least 6:1, at least 7:1,or at least 8; 1. In some embodiments, the ratio of the fusogenicpolypeptide to the binding polypeptide is between 1.5:1, 2:1, or 3:1, onthe low end of the range, and 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1on the high end of the range.

Activation Elements

Many of the methods and composition aspects of the present disclosureinclude an activation element, also referred to herein as a T cellactivation element, or a nucleic acid encoding an activation element.The restrictions associated with lentiviral (LV) transduction intoresting T cells are attributed to a series of pre-entry and post-entrybarriers as well as cellular restrictive factors (Strebel et al 2009.BMC Medicine 7:48). One restriction is the inability for the envelopepseudotyped-LV particles to recognize potential receptors and mediatefusion with the cellular membrane. However, under certain conditions,the transduction of resting T cells with HTV-1-based lentiviral vectorsis possible mostly upon T cell receptor (TCR) CD3 complex and CD28co-stimulation (Korin & Zack. 1998. Journal of Virology. 72:3161-8,Maurice et al. 2002. Blood 99:2342-50), as well as through exposure tocytokines (Cavalieri et al. 2003).

Cells of the immune system such as T lymphocytes recognize and interactwith specific antigens through receptors or receptor complexes which,upon recognition or an interaction with such antigens, cause activationof the cell and expansion in the body. An example of such a receptor isthe antigen-specific T lymphocyte receptor complex (TCR/CD3). The T cellreceptor (TCR) is expressed on the surface of T lymphocytes. Onecomponent, CD3, is responsible for intracellular signaling followingoccupancy of the TCR by ligand. The T lymphocyte receptor forantigen-CD3 complex (TCR/CD3) recognizes antigenic peptides that arepresented to it by the proteins of the major histocompatibility complex(MHC). Complexes of MHC and peptide are expressed on the surface ofantigen presenting cells and other T lymphocyte targets. Stimulation ofthe TCR/CD3 complex results in activation of the T lymphocyte and aconsequent antigen-specific immune response. The TCR/CD3 complex plays acentral role in the effector function and regulation of the immunesystem. Thus, activation elements provided herein, activate T cells bybinding to one or more components of the T cell receptor associatedcomplex, for example by binding to CD3. In some embodiments, theactivation element can activate alone. In other cases, the activationrequires activation through the TCR receptor complex in order to furtheractivate cells.

T lymphocytes also require a second, co-stimulatory signal to becomefully active in vivo. Without such a signal, T lymphocytes are eithernon-responsive to antigen binding to the TCR, or become anergic.However, the second, co-stimulatory signal is not required for thetransduction and expansion of T cells. Such a co-stimulatory signal, forexample, is provided by CD28, a T lymphocyte protein, which interactswith CD80 and CD86 on antigen-producing cells. As used herein, afunctional extracellular fragment of CD80 retains its ability tointeract with CD28. OX40, 4-1BB, and ICOS (Inducible COStimulator),other T lymphocyte proteins, and provides a co-stimulatory signal whenbound to one or more of its respective ligands: OX40L, 4-1BBL, andICOSLG.

Activation of the T cell receptor (TCR) CD3 complex and co-stimulationwith CD28 can occur by ex vivo exposure to solid surfaces (e.g. beads)coated with anti-CD3 and anti-CD28. In some embodiments of the methodsand compositions disclosed herein, resting T cells are activated byexposure to solid surfaces coated with anti-CD3 and anti-CD28 ex vivo.In other embodiments, resting T cells or NK cells, and in illustrativeembodiments resting T cells, are activated by exposure to solubleanti-CD3 antibodies (e.g. at 50-150, or 75-125, or 100 ng/ml). In suchembodiments, which can be part of methods for genetically modifying ortransducing, in illustrative embodiments without prior activation, suchactivation and/or contacting can be carried out by including anti-CD3 ina transduction reaction mixture and contacting with optional incubatingfor any of the times provided herein. Furthermore, such activation withsoluble anti-CD3 can occur by incubating lymphocytes, such as PBMCs, andin illustrative embodiments NK cells and in more illustrativeembodiments, T cells, after they are contacted with retroviral particlesin a media containing an anti-CD3. Such incubation can be for example,for between 5, 10, 15, 30, 45, 60, or 120 minutes on the low end of therange, and 15, 30, 45, 60, 120, 180, or 240 minutes on the high end ofthe range, for example, between 15 and 1 hours or 2 hours.

In certain illustrative embodiments of the methods and compositionsprovided herein, polypeptides that are capable of binding to anactivating T cell surface protein are presented as “activation elements”on the surface of replication incompetent recombinant retroviralparticles of the methods and compositions disclosed herein, which arealso aspects of the invention. In illustrative embodiments, theactivation elements on the surfaces of the replication incompetentrecombinant retroviral particles can include one or more polypeptidescapable of binding CD3. In illustrative embodiments, the activationelements on the surfaces of the replication incompetent recombinantretroviral particles can include one or more polypeptides capable ofbinding the epsilon chain of CD3 (CD3 epsilon). In other embodiments,the activation element on the surfaces of the replication incompetentrecombinant retroviral particles can include one or more polypeptidescapable of binding CD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81,and/or CD82 and optionally one or more polypeptides capable of bindingCD3. In illustrative embodiments, the activation element can be a T cellsurface protein agonist. The activation element can include apolypeptide that acts as a ligand for a T cell surface protein. In someembodiments, the polypeptide that acts as a ligand for a T cell surfaceprotein is, or includes, one or more of OX40L, 4-1BBL, or ICOSLG.

In some embodiments, one or typically more copies of one or more ofthese activation elements can be expressed on the surfaces of thereplication incompetent recombinant retroviral particles as polypeptidesseparate and distinct from the pseudotyping elements. In someembodiments, the activation elements can be expressed on the surfaces ofthe replication incompetent recombinant retroviral particles as fusionpolypeptides. In illustrative embodiments, the fusion polypeptidesinclude one or more activation elements and one or more pseudotypingelements. In further illustrative embodiments, the fusion polypeptideincludes anti-CD3, for example an anti-CD3scFv, or an anti-CD3scFvFc,and a viral envelope protein. In one example the fusion polypeptide isthe OKT-3scFv fused to the amino terminal end of a viral envelopeprotein such as the MuLV envelope protein, as shown in Maurice et al.(2002). In some embodiments, the fusion polypeptide is UCHT1scFv fusedto a viral envelope protein, for example the MuLV envelope protein (SEQID NO:341), the MuLVSUx envelope protein (SEQ ID NO:454), VSV-G (SEQ IDNO:455 or SEQ ID NO:456), or functional variants or fragments thereof,including any of the membrane protein truncations provided herein. Insuch fusion constructs, and any other constructs wherein an activationelement is tethered to the surface of a retroviral particle,illustrative embodiments especially for compositions and methods hereinfor transducing lymphocytes in whole blood, do not include any bloodprotein (e.g. blood Factor (e.g. Factor X)) cleavage sites in theportion of the fusion protein that resides outside the retroviralparticle. In some embodiments, the fusion constructs do not include anyfurin cleavage sites. Furin is a membrane bound protease expressed inall mammalian cells examined, some of which is secreted and active inblood plasma (See e.g. C. Fernandez et al. J. Internal. Medicine (2018)284; 377-387). Mutations can be made to fusion constructs using knownmethods to remove such protease cleavage sites.

Polypeptides that bind CD3, CD28, OX40, 4-1BB, or ICOS are referred toas activation elements because of their ability to activate resting Tcells. In certain embodiments, nucleic acids encoding such an activatingelement are found in the genome of a replication incompetent recombinantretroviral particle that contains the activating element on its surface.In other embodiments, nucleic acids encoding an activating element arenot found in the replication incompetent recombinant retroviral particlegenome. In still other embodiments, the nucleic acids encoding anactivating element are found in the genome of a virus packaging cell.

In some embodiments, the activation element is a polypeptide capable ofbinding to CD3. In certain embodiments the polypeptide capable ofbinding to CD3, binds to CD3D, CD3E, CD3G, or CD3Z. In illustrativeembodiments the activation element is a polypeptide capable of bindingto CD3E. In some embodiments, the polypeptide capable of binding to CD3is an anti-CD3 antibody, or a fragment thereof that retains the abilityto bind to CD3. In illustrative embodiments, the anti-CD3 antibody orfragment thereof is a single chain anti-CD3 antibody, such as but notlimited to, an anti-CD3 scFv. In another illustrative embodiment, thepolypeptide capable of binding to CD3 is anti-CD3scFvFc.

A number of anti-human CD3 monoclonal antibodies and antibody fragmentsthereof are available, and can be used in the present invention,including but not limited to UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66,WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8,T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01.

In some embodiments, the activation element is a polypeptide capable ofbinding to CD28. In some embodiments, the polypeptide capable of bindingto CD28 is an anti-CD28 antibody, or a fragment thereof that retains theability to bind to CD28. In other embodiments, the polypeptide capableof binding to CD28 is CD80, CD86, or a functional fragment thereof thatis capable of binding CD28 and inducing CD28-mediated activation of Akt,such as an external fragment of CD80. In some aspects herein, anexternal fragment of CD80 means a fragment that is typically present onthe outside of a cell in the normal cellular location of CD80, thatretains the ability to bind to CD28. In illustrative embodiments, theanti-CD28 antibody or fragment thereof is a single chain anti-CD28antibody, such as, but not limited to, an anti-CD28 scFv. In anotherillustrative embodiment, the polypeptide capable of binding to CD28 isCD80, or a fragment of CD80 such as an external fragment of CD80.

Anti-CD28 antibodies are known in the art and can include, asnon-limiting examples, monoclonal antibody 9.3, an IgG2a antibody (Dr.Jeffery Ledbetter. Bristol Myers Squibb Corporation, Seattle, Wash.),monoclonal antibody KOLT-2, an IgG1 antibody, 15E8, an IgG1 antibody,248.23.2, an IgM antibody and EX5.3D10, an IgG2a antibody.

In an illustrative embodiment, an activation element includes twopolypeptides, a polypeptide capable of binding to CD3 and a polypeptidecapable of binding to CD28.

In certain embodiments, tire polypeptide capable of binding to CD3 orCD28 is an antibody, a single chain monoclonal antibody or an antibodyfragment, for example a single chain antibody fragment. Accordingly, theantibody fragment can be, for example, a single chain fragment variableregion (scFv), an antibody binding (Fab) fragment of an antibody, asingle chain antigen-binding fragment (scFab), a single chainantigen-binding fragment without cysteines (scFabΔC), a fragmentvariable region (Fv), a construct specific to adjacent epitopes of anantigen (CRAb), or a single domain antibody (VH or VL).

In any of the embodiments disclosed herein, an activation element, or anucleic acid encoding tire same, can include a dimerizing or higherorder multimerizing motif. Dimerizing and multimerizing motifs arewell-known in tire art and a skilled artisan will understand how toincorporate them into tire polypeptides for effective dimerization ormultimerization. For example, in some embodiments, tire activationelement that includes a dimerizing motif can be one or more polypeptidescapable of binding to CD3 and/or CD28. In some embodiments, thepolypeptide capable of binding to CD3 is an anti-CD3 antibody, or afragment thereof that retains the ability to bind to CD3. Inillustrative embodiments, the anti-CD3 antibody or fragment thereof is asingle chain anti-CD3 antibody, such as but not limited to, an anti-CD3scFv. In another illustrative embodiment, the polypeptide capable ofbinding to CD3 is anti-CD3scFvFc, which in some embodiments isconsidered an anti-CD3 with a dimerizing motif without any additionaldimerizing motif, since anti-CD3scFvFc constructs are known to becapable of dimerizing without the need for a separate dimerizing motif.

In some embodiments, the dimerizing or multimerizing motif, or a nucleicacid sequence encoding the same, can be an amino acid sequence fromtransmembrane polypeptides that naturally exist as homodimers ormultimers. In some embodiments, the dimerizing or multimerizing motif,or a nucleic acid sequence encoding the same, can be an amino acidsequence from a fragment of a natural protein or an engineered protein.In one embodiment, the homodimeric polypeptide is a leucine zippermotif-containing polypeptide (leucine zipper polypeptide). For example,a leucine zipper polypeptide derived from c-JUN, non-limiting examplesof which are disclosed related to chimeric lymphoproliferative elements(CLEs) herein.

In some embodiments, these transmembrane homodimeric polypeptides caninclude early activation antigen CD69 (CD69), Transferrin receptorprotein 1 (CD71), B-cell differentiation antigen (CD72), T-cell surfaceprotein tactile (CD96), Endoglin (Cd105), Killer cell lectin-likereceptor subfamily B member 1 (Cd161), P-selectin glycoprotein ligand 1(Cd162), Glutamyl aminopeptidase (Cd249), Tumor necrosis factor receptorsuperfamily member 16 (CD271), Cadherin-1 (E-Cadherin) (Cd324), oractive fragments thereof. In some embodiments, the dimerizing motif, andnucleic acid encoding the same, can include an amino acid sequence fromtransmembrane proteins that dimerize upon ligand (also referred toherein as a dimerizer or dimerizing agent) binding. In some embodiments,the dimerizing motif and dimerizer can include (where the dimerizer isin parentheses following the dimerizer-binding pair): FKBP and FKBP(rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate);or DmrB and DmrB (AP20187). As noted above, rapamycin can serve as adimerizer. Alternatively, a rapamycin derivative or analog can be used(see, e.g., WO96/41865; WO 99/36553; WO 01/14387; and Ye et al (1999)Science 283:88-91). For example, analogs, homologs, derivatives, andother compounds related structurally to rapamycin (“rapalogs”) include,among others, variants of rapamycin having one or more of the followingmodifications relative to rapamycin: demethylation, elimination orreplacement of the methoxy at C7, C42 and/or C29; elimination,derivatization or replacement of the hydroxy at C13, C43 and/or C28;reduction, elimination or derivatization of the ketone at C14, C24and/or C30; replacement of the 6-membered pipecolate ring with a5-membered prolyl ring; and alternative substitution on the cyclohexylring or replacement of the cyclohexyl ring with a substitutedcyclopentyl ring. Additional information is presented in, e.g., U.S.Pat. Nos. 5,525,610; 5,310,903 5,362,718; and 5,527,907. Selectiveepimerization of tire C-28 hydroxyl group has been described (see, e.g.,WO 01/14387). Additional synthetic dimerizing agents suitable for use asan alternative to rapamycin include those described in U.S. PatentPublication No. 2012/0130076. As noted above, coumermycin can serve as adimerizing agent. Alternatively, a coumermycin analog can be used (see,e.g., Farrar et al. (1996) Nature 383:178-181; and U.S. Pat. No.6,916,846). As noted above, in some cases, the dimerizing agent ismethotrexate, e.g., a non-cytotoxic, homo-bifunctional methotrexatedimer (see, e.g., U.S. Pat. No. 8,236,925). Although some embodiments oflymphoproliferative elements include a dimerizing agent, in some aspectsand illustrative embodiments, a lymphoproliferative element isconstitutively active, and is other than a lymphoproliferative elementthat requires a dimerizing agent for activation.

In some embodiments, when present on the surface of replicationincompetent recombinant retroviral particles, an activation elementincluding a dimerizing motif can be active in the absence of adimerizing agent. For example, activation elements including adimerizing motif from transmembrane homodimeric polypeptides includingCD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, Cd324, activemutants thereof, and/or active fragments thereof can be active in theabsence a dimerizing agent. In some embodiments, the activation elementcan be an anti-CD3 single chain fragment and include a dimerizing motifselected from the group consisting of CD69, CD71, CD72, CD96, Cd105,Cd161, Cd16, Cd249, CD271, Cd324, active mutants thereof, and/or activefragments thereof.

In some embodiments, when present on the surface of replicationincompetent recombinant retroviral particles, an activation elementincluding a dimerizing motif can be active in the presence of adimerizing agent. For example, activation elements including adimerizing motif from FKBP, GyrB, DHFR, or DmrB can be active in thepresence of the respective dimerizing agents or analogs thereof, e.g.rapamycin, coumermycin, methotrexate, and AP20187, respectively. In someembodiments, the activation element can be a single chain antibodyfragment against anti-CD3 or anti-CD28, or another molecule that bindsCD3 or CD28, and the dimerizing motif and dimerizing agent can beselected from the group consisting of FKBP and rapamycin or analogsthereof, GyrB and coumermycin or analogs thereof, DHFR and methotrexateor analogs thereof, or DmrB and AP20187 or analogs thereof.

In some embodiments, an activation element is fused to a heterologoussignal sequence and/or a heterologous membrane attachment sequence or amembrane bound protein, all of which help direct the activation elementto the membrane. The heterologous signal sequence targets the activationelement to the endoplasmic reticulum, where the heterologous membraneattachment sequence covalently attaches to one or several fatty acids(also known as posttranslational lipid modification) such that theactivation elements that are fused to the heterologous membraneattachment sequence are anchored in the lipid rafts of the plasmamembrane. In some embodiments, posttranslational lipid modification canoccur via myristoylation, palmitoylation, or GPI anchorage.Myristoylation is a post-translational protein modification whichcorresponds to the covalent linkage of a 14-carbon saturated fatty acid,the myristic acid, to the N-terminal glycine of a eukaryotic or viralprotein. Palmitoylation is a post-translational protein modificationwhich corresponds to the covalent linkage of a C16 acyl chain tocysteines, and less frequently to serine and threonine residues, ofproteins. GPI anchorage refers to the attachment ofglycosylphosphatidylinositol, or GPI, to the C-term in us of a proteinduring posttranslational modification.

In some embodiments, the heterologous membrane attachment sequence is aGPI anchor attachment sequence. The heterologous GPI anchor attachmentsequence can be derived from any known GPI-anchored protein (reviewed inFerguson M A J, Kinoshita T, Hart G W. GlycosylphosphatidylinositolAnchors. In: Varki A, Cummings R D, Esko J D, et al., editors.Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (N.Y.): ColdSpring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments,the heterologous GPI anchor attachment sequence is the GPI anchorattachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, andCD87. In some embodiments, the heterologous GPI anchor attachmentsequence is derived from CD16. In illustrative embodiments, theheterologous GPI anchor attachment sequence is derived from Fc receptorFcγRIIIb (CD16b) or decay accelerating factor (DAF), otherwise known ascomplement decay-accelerating factor or CD55.

In some embodiments, one or both of the activation elements include aheterologous signal sequence to help direct expression of the activationelement to the cell membrane. Any signal sequence that is active in thepackaging cell line can be used. In some embodiments, the signalsequence is a DAF signal sequence. In illustrative embodiments, anactivation element is fused to a DAF signal sequence at its N terminusand a GPI anchor attachment sequence at its C terminus.

In an illustrative embodiment, the activation element includes anti-CD3scFvFc fused to a GPI anchor attachment sequence derived from CD14 andCD80 fused to a GPI anchor attachment sequence derived from CD16b; andboth are expressed on the surface of a replication incompetentrecombinant retroviral particle provided herein. In some embodiments,the anti-CD3 scFvFc is fused to a DAF signal sequence at its N terminusand a GPI anchor attachment sequence derived from CD14 at its C terminusand the CD80 is fused to a DAF signal sequence at its N terminus and aGPI anchor attachment sequence derived from CD16b at its C terminus; andboth are expressed on the surface of a replication incompetentrecombinant retroviral particle provided herein. In some embodiments,tire DAF signal sequence includes amino acid residues 1-30 of the DAFprotein.

Membrane-Bound Cytokines

Some embodiments of the method and composition aspects provided herein,include a membrane-bound cytokine, or polynucleotides encoding amembrane-bound cytokine. Cytokines are typically, but not always,secreted proteins. Cytokines that are naturally secreted can beengineered as fusion proteins to be membrane-bound. Membrane-boundcytokine fusion polypeptides are included in methods and compositionsdisclosed herein, and are also an aspect of the invention. In someembodiments, replication incompetent recombinant retroviral particleshave a membrane-bound cytokine fusion polypeptide on their surface thatis capable of binding a T cell and/or NK cell and promotingproliferation and/or survival thereof. Typically, membrane-boundpolypeptides are incorporated into the membranes of replicationincompetent recombinant retroviral particles, and when a cell istransduced by the replication incompetent recombinant retroviralparticles, the fusion of the retroviral and host cell membranes resultsin the polypeptide being bound to the membrane of the transduced cell.

In some embodiments, the cytokine fusion polypeptide includes IL-2,IL-7, IL-15, or an active fragment thereof. The membrane-bound cytokinefusion polypeptides are typically a cytokine fused to heterologoussignal sequence and/or a heterologous membrane attachment sequence. Insome embodiments, the heterologous membrane attachment sequence is a GPIanchor attachment sequence. The heterologous GPI anchor attachmentsequence can be derived from any known GPI-anchored protein (reviewed inFerguson M A J, Kinoshita T, Hart G W. GlycosylphosphatidylinositolAnchors. In: Varki A, Cummings R D, Esko J D, et al., editors.Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (N.Y.): ColdSpring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments,the heterologous GPI anchor attachment sequence is the GPI anchorattachment sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80, andCD87. In some embodiments, the heterologous GPI anchor attachmentsequence is derived from CD16. In an illustrative embodiment, theheterologous GPI anchor attachment sequence is derived from Fc receptorFcγRIIIb (CD16b). In some embodiments, tire GPI anchor is the GPI anchorof DAF.

In illustrative embodiments, the membrane-bound cytokine is a fusionpolypeptide of a cytokine fused to DAF. DAF is known to accumulate inlipid rafts that are incorporated into the membranes of replicationincompetent recombinant retroviral particles budding from packagingcells. Accordingly, not to be limited by theory, it is believed that DAFfusion proteins are preferentially targeted to portions of membranes ofpackaging cells that will become part of a recombinant retroviralmembrane.

In non-limiting illustrative embodiments, the cytokine fusionpolypeptide is an IL-7, or an active fragment thereof, fused to DAF. Ina specific non-limiting illustrative embodiment, the fusion cytokinepolypeptide includes in order: the DAF signal sequence (residues 1-31 ofDAF), IL-7 without its signal sequence, and residues 36-525 of DAF.

Packaging Cell Lines/Methods of Making Recombinant Retroviral Particles

The present disclosure provides mammalian packaging cells and packagingcell lines that produce replication incompetent recombinant retroviralparticles. The cell lines that produce replication incompetentrecombinant retroviral particles are also referred to herein aspackaging cell lines. A non-limiting example of such method isillustrated in WO2019/055946. Further exemplary methods for makingretroviral particles are provided herein, for example in the Examplessection herein. Such methods include, for example, a 4 plasmid system ora 5 plasmid system when a nucleic acid encoding an additional membranebound protein, such as a T cell activation element that is not a fusionwith the viral envelope, such as a GPI-linked anti-CD3, is included (SeeWO2019/05546). In an illustrative embodiment, provided herein is a 4plasmid system in which a T cell activation element, such as aGPI-linked anti-CD3, is encoded on one of the packaging plasmids such asthe plasmid encoding the viral envelope or the plasmid encoding REV, andoptionally a second viral membrane-associated transgene such as amembrane bound cytokine can be encoded on the other packaging plasmid.In each case the nucleic acid encoding the viral protein is separatedfrom the transgene by an IRES or a ribosomal skip sequence such as P2Aor T2A. Such 4 plasmid system and associated polynucleotides as statedin the Examples, provided increased titers as compared to a 5 vectorsystem in transient transfections, and thus provide illustrativeembodiments herein. The present disclosure provides packaging cells andmammalian cell lines that are packaging cell lines that producereplication incompetent recombinant retroviral particles thatgenetically modify target mammalian cells and the target mammalian cellsthemselves. In illustrative embodiments, the packaging cell comprisesnucleic acid sequences encoding a packageable RNA genome of thereplication incompetent retroviral particle, a REV protein, a gagpolypeptide, a pol polypeptide, and a pseudotyping element.

The cells of the packaging cell line can be adherent or suspensioncells. Exemplary cell types are provided hereinbelow. In illustrativeembodiments, the packaging cell line can be a suspension cell line, i.e.a cell line that does not adhere to a surface during growth. The cellscan be grown in a chemically-defined media and/or a scrum-free media. Insome embodiments, the packaging cell line can be a suspension cell linederived from an adherent cell line, for example, the HEK293 cell linecan be grown in conditions to generate a suspension-adapted HEK293 cellline according to methods known in the art. The packaging cell line istypically grown in a chemically defined media. In some embodiments, thepackaging cell line media can include serum. In some embodiments, thepackaging cell line media can include a serum replacement, as known inthe art. In illustrative embodiments, the packaging cell line media canbe serum-free media. Such media can be a chemically defined, serum-freeformulation manufactured in compliance with Current Good ManufacturingPractice (CGMP) regulations of the US Food and Drug Administration(FDA). The packaging cell line media can be xeno-finee (free ofnon-human animal media proteins and other components) and complete. Insome embodiments, the packaging cell line media has been cleared byregulatory agencies for use in ex vivo cell processing, such as an FDA510(k) cleared device.

Accordingly, in one aspect, provided herein is a method of making areplication incompetent recombinant retroviral particle including: A.culturing a packaging cell in suspension in serum-free media, whereinthe packaging cell comprises nucleic acid sequences encoding apackageable RNA genome of the replication incompetent retroviralparticle, a REV protein, a gag polypeptide, a pol polypeptide, and apseudotyping element; and B. harvesting the replication incompetentrecombinant retroviral particle from the serum-free media. In anotheraspect provided herein is a method of transducing a lymphocyte with areplication incompetent recombinant retroviral particle comprising: A.culturing a packaging cell in suspension in serum-free media, whereinthe packaging cell comprises nucleic acid sequences encoding apackageable RNA genome of the replication incompetent retroviralparticle, a REV protein, a gag polypeptide, a pol polypeptide, and apseudotyping element; B. harvesting the replication incompetentrecombinant retroviral particle from the serum-free media; and C.contacting the lymphocyte with the replication incompetent recombinantretroviral particle, wherein the contacting is performed for less than24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1hour, 30 minutes, or 15 minutes (or between contacting and noincubation, or 15 minutes, 30 minutes, 1, 2, 3, or 4 hours on the lowend of the range and 1, 2, 3, 4, 6, 8, 12, 18, 20, or 24 hours on thehigh end of the range), thereby transducing the lymphocyte.

The packageable RNA genome, in certain illustrative embodiments, isdesigned to express one or more target polypeptides, including as anon-limiting example, any of the engineered signaling polypeptidesdisclosed herein and/or one or more (e.g. two or more) inhibitory RNAmolecules in opposite orientation (e.g., encoding on the opposite strandand in the opposite orientation), from retroviral components such as gagand pol. For example, the packageable RNA genome can include from 5′ to3′; a 5′ long terminal repeat, or active truncated fragment thereof; anucleic acid sequence encoding a retroviral cis-acting RNA packagingelement; a nucleic acid sequence encoding a first and optionally secondtarget polypeptide, such as, but not limited to, an engineered signalingpolypeptide(s) in opposite orientation, which can be driven off apromoter in this opposite orientation with respect to the 5′ longterminal repeat and the cis-acting RNA packaging element, which in someembodiments is called a “fourth” promoter for convenience only (andsometimes referred to herein as the promoter active in T cells and/or NKcells), which is active in a target cell such as a T cell and/or an NKcell but in illustrative examples is not active in the packaging cell oris only inducibly or minimally active in the packaging cell; and a 3′long terminal repeat, or active truncated fragment thereof. In someembodiments, the packageable RNA genome can include a central polypurinetract (cPPT)/central termination sequence (CTS) element. In someembodiments, the retroviral cis-acting RNA packaging element can be HIVPsi. In some embodiments, the retroviral cis-acting RNA packagingelement can be the Rev Response Element. The engineered signalingpolypeptide driven by the promoter in the opposite orientation from the5′ long terminal repeat, in illustrative embodiments, is one or more ofthe engineered signaling polypeptides disclosed herein and canoptionally express one or more inhibitory RNA molecules as disclosed inmore detail herein and in WO2017/165245A2, WO2018/009923A1, andWO2018/161064A1.

It will be understood that promoter number, such as a first, second,third, fourth, etc. promoter is for convenience only. A promoter that iscalled a “fourth” promoter should not be taken to imply that there areany additional promoters, such as first, second or third promoters,unless such other promoters are explicitly recited. It should be notedthat each of the promoters are capable of driving expression of atranscript in an appropriate cell type and such transcript forms atranscription unit.

In some embodiments, the engineered signaling polypeptide can include afirst lymphoproliferative element. Suitable lymphoproliferative elementsare disclosed in other sections herein. As a non-limiting example, thelymphoproliferative element can be expressed as a fusion with arecognition domain, such as an eTag, as disclosed herein. In someembodiments, the packageable RNA genome can further include a nucleicacid sequence encoding a second engineered polypeptide including achimeric antigen receptor, encoding any CAR embodiment provided herein.For example, the second engineered polypeptide can include a firstantigen-specific targeting region, a first transmembrane domain, and afirst intracellular activating domain. Examples of antigen-specifictargeting regions, transmembrane domains, and intracellular activatingdomains are disclosed elsewhere herein. In some embodiments where thetarget cell is a T cell, the promoter that is active in a target cell isactive in a T cell, as disclosed elsewhere herein.

In some embodiments, the engineered signaling polypeptide can include aCAR, and tire nucleic acid sequence can encode any CAR embodimentprovided herein. For example, the engineered polypeptide can include afirst antigen-specific targeting region, a first transmembrane domain,and a first intracellular activating domain. Examples ofantigen-specific targeting regions, transmembrane domains, andintracellular activating domains are disclosed elsewhere herein. In someembodiments, the packageable RNA genome can further include a nucleicacid sequence encoding a second engineered polypeptide. In someembodiments, the second engineered polypeptide can be alymphoproliferative element. In some embodiments where the target cellis a T cell or NK cell, the promoter that is active in a target cell isactive in a T cell or NK cell, as disclosed elsewhere herein.

In some embodiments, the packageable RNA genome included in any of tireaspects provided herein, can further include a riboswitch, as discussedin WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1. In someembodiments, the nucleic acid sequence encoding the engineered signalingpolypeptide can be in a reverse orientation with respect to the 5′ to 3′orientation established by the 5′ LTR and the 3′ LTR. In furtherembodiments, the packageable RNA genome can further include a riboswitchand, optionally, the riboswitch can be in reverse orientation. In any ofthe embodiments disclosed herein, a polynucleotide including any of theelements can include a primer binding site. In illustrative embodiments,insulators and/or polyadenylation sequences can be placed before, after,between, or near genes to prevent or reduce unregulated transcription.In some embodiments, the insulator can be chicken HS4 insulator, Kaisoinsulator, SAR/MAR elements, chimeric chicken insulator-SAR elements,CTCF insulator, the gypsy insulator, or the β-globin insulator orfragments thereof known in the art. In some embodiments, the insulatorand/or polyadenylation sequence can be hGH polyA (SEQ ID NO:316), SPA1(SEQ ID NO:317), SPA2 (SEQ ID NO:318), b-globin polyA spacer B (SEQ IDNO:319), b-globin polyA spacer A (SEQ ID NO:320), 250 cHS4 insulator v1(SEQ ID NO:321), 250 cHS4 insulator v2 (SEQ ID NO:322), 650 cHS4insulator (SEQ ID NO:323), 400 cHS4 insulator (SEQ ID NO:324), 650 cHS4insulator and b-globin polyA spacer B (SEQ ID NO:325), or b-globin polyAspacer B and 650 cHS4 insulator (SEQ ID NO:326).

In any of the embodiments disclosed herein, a nucleic acid sequenceencoding Vpx can be on the second or an optional third transcriptionalunit, or on an additional transcriptional unit that is operably linkedto the first inducible promoter.

Some aspects of the present disclosure include or are cells, inillustrative examples, mammalian cells, that are used as packaging cellsto make replication incompetent recombinant retroviral particles, suchas lentiviruses, for transduction of T cells and/or NK cells.

Any of a wide variety of cells can be selected for in vitro productionof a virus or virus particle, such as a redirected recombinantretroviral particle, according to the invention. Eukaryotic cells aretypically used, particularly mammalian cells including human, simian,canine, feline, equine and rodent cells. In illustrative examples, thecells are human cells. In further illustrative embodiments, the cellsreproduce indefinitely, and are therefore immortal. Examples of cellsthat can be advantageously used in the present invention include NIH 3T3cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293Tcells and any cells derived from such cells, such as gpnlslacZ φNXcells, which are derived from 293T cells. Highly transfectable cells,such as human embryonic kidney 293T cells, can be used. By “highlytransfectable” it is meant that at least about 50%, more preferably atleast about 70% and most preferably at least about 80% of the cells canexpress the genes of the introduced DNA.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No CCLlO), PC 12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATl cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, and the like

Retroviral Genome Size

In the methods and compositions provided herein, the recombinantretroviral genomes, in non-limiting illustrative examples, lentiviralgenomes, have a limitation to the number of polynucleotides that can bepackaged into the viral particle. In some embodiments provided herein,the polypeptides encoded by the polynucleotide encoding region can betruncations or other deletions that retain a functional activity-suchthat the polynucleotide encoding region is encoded by less nucleotidesthan the poly-nucleotide encoding region for the wild-type poly-peptide.In some embodiments, the polypeptides encoded by the poly-nucleotideencoding region can be fusion polypeptides that can be expressed fromone promoter. In some embodiments, the fusion poly-peptide can have acleavage signal to generate two or more functional poly-peptides fromone fusion polypeptide and one promoter. Furthermore, some functionsthat are not required after initial ex vivo transduction are notincluded in the retroviral genome, but rather are present on the surfaceof the replication incompetent recombinant retroviral particles via thepackaging cell membrane. These various strategies are used herein tomaximize the functional elements that are packaged within thereplication incompetent recombinant retroviral particles.

In some embodiments, the recombinant retroviral genome to be packagedcan be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and8,000 nucleotides on the low end of the range and 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides on thehigh end of the range. The retroviral genome to be packaged includes oneor more polynucleotide regions encoding a first and second engineeringsignaling polypeptide as disclosed in detail herein. In someembodiments, the recombinant retroviral genome to be packaged can beless than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000nucleotides. Functions discussed elsewhere herein that can be packagedinclude required retroviral sequences for retroviral assembly andpackaging, such as a retroviral rev, gag, and pol coding regions, aswell as a 5′ LTR and a 3′ LTR, or an active truncated fragment thereof,a nucleic acid sequence encoding a retroviral cis-acting RNA packagingelement and a cPPT/CTS element. Furthermore, in illustrative embodimentsa replication incompetent recombinant retroviral particle herein caninclude any one or more or all of the following, in some embodiments inreverse orientation with respect to a 5′ to 3′ orientation establishedby the retroviral 5′ LTR and 3′ LTR (as illustrated in WO2019/055946 asa non-limiting example): one or more polynucleotide regions encoding afirst and second engineering signaling polypeptide, at least one ofwhich includes at least one lymphoproliferative element; a secondengineered signaling polypeptide that can include a chimeric antigenreceptor; an miRNA, a control element, such as a riboswitch, whichtypically regulates expression of the first and/or the secondengineering signaling polypeptide; a recognition domain, an intron, apromoter that is active in a target cell, such as a T cell, a 2Acleavage signal and/or an IRES.

Recombinant Retroviral Particles

Recombinant retroviral particles are disclosed in methods andcompositions provided herein, for example, to transduce T cells and/orNK cells to make genetically modified T cells and/or NK cells. Therecombinant retroviral particles are themselves aspects of the presentinvention. Typically, the recombinant retroviral particles included inaspects provided herein, are replication incompetent, meaning that arecombinant retroviral particle cannot replicate once it leaves thepackaging cell. In illustrative embodiments, the recombinant retroviralparticles are lentiviral particles.

Provided herein in some aspects are replication incompetent recombinantretroviral particles for use in transducing cells, typically lymphocytesand illustrative embodiments T cells and/or NK cells. The replicationincompetent recombinant retroviral particles can include any of thepseudotyping elements discussed elsewhere herein. In some embodiments,the replication incompetent recombinant retroviral particles can includeany of the activation elements discussed elsewhere herein. In oneaspect, provided herein is a replication incompetent recombinantretroviral particle including a polynucleotide including: A. one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode achimeric antigen receptor (CAR); and B. a pseudotyping element and a Tcell activation element on its surface, wherein the T cell activationelement is not encoded by a polynucleotide in the replicationincompetent recombinant retroviral particle. In some embodiments, the Tcell activation element can be any of the activation elements discussedelsewhere herein. In illustrative embodiments, the T cell activationelement can be anti-CD3 scFvFc. In another aspect, provided herein is areplication incompetent recombinant retroviral particle, including apolynucleotide including one or more transcriptional units operativelylinked to a promoter active in T cells and/or NK cells, wherein the oneor more transcriptional units encode a first polypeptide including achimeric antigen receptor (CAR) and a second polypeptide including alymphoproliferative element. In some embodiments, thelymphoproliferative element can be a chimeric lymphoproliferativeelement. In illustrative embodiments, the lymphoproliferative elementdoes not comprise IL-7 tethered to the IL-7 receptor alpha chain or afragment thereof. In some embodiments the lymphoproliferative elementdoes not comprise IL-15 tethered to the IL-2/IL-15 receptor beta chain.

In some aspects, provided herein is a replication incompetentrecombinant retroviral particle, comprising a polynucleotide comprisingone or more transcriptional units operatively linked to a promoteractive in T cells and/or NK cells, wherein the one or moretranscriptional units encode a first polypeptide comprising a chimericantigen receptor (CAR) and a second polypeptide comprising a chimericlymphoproliferative element, for example a constitutively activechimeric lymphoproliferative element. In illustrative embodiments, thechimeric lymphoproliferative element does not comprise a cytokinetethered to its cognate receptor or tethered to a fragment of itscognate receptor.

Provided herein in some aspects, is a recombinant retroviral particlethat includes (i) a pseudotyping element capable of binding to a T celland/or NK cell and facilitating membrane fusion of the recombinantretroviral particle thereto; (ii) a polynucleotide having one or moretranscriptional units operatively linked to a promoter active in T cellsand/or NK cells, wherein the one or more transcriptional units encode afirst engineered signaling polypeptide having a chimeric antigenreceptor that includes an antigen-specific targeting region, atransmembrane domain, and an intracellular activating domain, and asecond engineered signaling polypeptide that includes at least onelymphoproliferative element; wherein expression of the first engineeredsignaling polypeptide and/or the second engineered signaling polypeptideare regulated by an in vivo control element; and (iii) an activationelement on its surface, wherein the activation element is capable ofbinding to a T cell and/or NK cell and is not encoded by apolynucleotide in the recombinant retroviral particle. In someembodiments, the promoter active in T cells and/or NK cells is notactive in the packaging cell line or is only active in the packagingcell line in an inducible manner. In any of the embodiments disclosedherein, either of the first and second engineered signaling polypeptidescan have a chimeric antigen receptor and the other engineered signalingpolypeptide can have at least one lymphoproliferative element.

Various elements and combinations of elements that are included inreplication incompetent, recombinant retroviral particles are providedthroughout this disclosure, such as, for example, pseudotyping elements,activation elements, and membrane bound cytokines, as well as nucleicacid sequences that are included in a genome of a replicationincompetent, recombinant retroviral particle such as, but not limitedto, a nucleic acid encoding a CAR; a nucleic acid encoding alymphoproliferative element; a nucleic acid encoding a control element,such as a riboswitch; a promoter, especially a promoter that isconstitutively active or inducible in a T cell; and a nucleic acidencoding an inhibitory RNA molecule. Furthermore, various aspectsprovided herein, such as methods of making recombinant retroviralparticles, methods for performing adoptive cell therapy, and methods fortransducing T cells, produce and/or include replication incompetent,recombinant retroviral particles. Replication incompetent recombinantretroviruses that are produced and/or included in such methodsthemselves form separate aspects of the present invention as replicationincompetent, recombinant retroviral particle compositions, which can bein an isolated form. Such compositions can be in dried down (e.g.lyophilized) form or can be in a suitable solution or medium known inthe art for storage and use of retroviral particles.

Accordingly, as a non-limiting example, provided herein in anotheraspect, is a replication incompetent recombinant retroviral particlehaving in its genome a polynucleotide having one or more nucleic acidsequences operatively linked to a promoter active in T cells and/or NKcells that in some instances, includes a first nucleic acid sequencethat encodes one or more (e.g. two or more) inhibitory RNA moleculesdirected against one or more RNA targets and a second nucleic acidsequence that encodes a chimeric antigen receptor, or CAR, as describedherein. In other embodiments, a third nucleic acid sequence is presentthat encodes at least one lymphoproliferative element describedpreviously herein that is not an inhibitory RNA molecule. In certainembodiments, the polynucleotide includes one or more riboswitches aspresented herein, operably linked to the first nucleic acid sequence,the second nucleic acid sequence, and/or the third nucleic acidsequence, if present. In such a construct, expression of one or moreinhibitory RNAs, the CAR, and/or one or more lymphoproliferativeelements that are not inhibitory RNAs is controlled by the riboswitch.In some embodiments, two to 10 inhibitory RNA molecules are encoded bythe first nucleic acid sequence. In further embodiments, two to sixinhibitory RNA molecules are encoded by the first nucleic acid sequence.In illustrative embodiments, 4 inhibitory RNA molecules are encoded bythe first nucleic acid sequence. In some embodiments, the first nucleicacid sequence encodes one or more inhibitory RNA molecules and islocated within an intron. In certain embodiments, the intron includesall or a portion of a promoter. The promoter can be a Pol I, Pol II, orPol III promoter. In some illustrative embodiments, the promoter is aPol II promoter. In some embodiments, the intron is adjacent to anddownstream of the promoter active in a T cell and/or NK cell. In someembodiments, the intron is EF1-α intron A.

Recombinant retroviral particle embodiments herein include those whereinthe retroviral particle comprises a genome that includes one or morenucleic acids encoding one or more inhibitory RNA molecules. Variousalternative embodiments of such nucleic acids that encode inhibitory RNAmolecules that can be included in a genome of a retroviral particle,including combinations of such nucleic acids with other nucleic acidsthat encode a CAR or a lymphoproliferative element other than aninhibitory RNA molecule, are included for example, in the inhibitory RNAsection provided herein, as well as in various other paragraphs thatcombine these embodiments. Furthermore, various alternatives of suchreplication incompetent recombinant retroviruses can be identified byexemplary nucleic acids that are disclosed within packaging cell lineaspects disclosed herein. A skilled artisan will recognize thatdisclosure in this section of a recombinant retroviral particle thatincludes a genome that encodes one or more (e.g. two or more) inhibitoryRNA molecules, can be combined with various alternatives for suchnucleic acids encoding inhibitory RNA molecules provided in othersections herein. Furthermore, a skilled artisan will recognize that suchnucleic acids encoding one or more inhibitory RNA molecules can becombined with various other functional nucleic acid elements providedherein, as for example, disclosed in the section herein that focuses oninhibitory RNA molecules and nucleic acid encoding these molecules. Inaddition, the various embodiments of specific inhibitory RNA moleculesprovided herein in other sections can be used in recombinant retroviralparticle aspects of the present disclosure.

Necessary elements of recombinant retroviral vectors, such as lentiviralvectors, are known in the art. These elements are included in thepackaging cell line section and in details for making replicationincompetent, recombinant retroviral particles provided in the Examplessection and as illustrated in WO2019/055946. For example, lentiviralparticles typically include packaging elements REV, GAG and POL, whichcan be delivered to packaging cell lines via one or more packagingplasmids, a pseudotyping element, various examples which are providedherein, which can be delivered to a packaging cell line via apseudotyping plasmid, and a genome, which is produced by apolynucleotide that is delivered to a host cell via a transfer plasmid.This polynucleotide typically includes the viral LTRs and a psipackaging signal. The 5′ LTR can be a chimeric 5′ LTR fused to aheterologous promoter, which includes 5′ LTRs that are not dependent onTat transactivation. The transfer plasmid can be self-inactivating, forexample, by removing a U3 region of the 3′ LTR. In some non-limitingembodiments, Vpu, such as a polypeptide comprising Vpu (sometimes calleda “Vpu polypeptide” herein) including but not limited to, Src-FLAG-Vpu,is packaged within the retroviral particle for any composition or methodaspect and embodiment provided herein that includes a retroviralparticle. In some non-limiting embodiments, Vpx, such as Src-FLAG-Vpx,is packaged within the retroviral particle. Not to be limited by theory,upon transduction of a T cells, Vpx enters the cytosol of the cells andpromotes the degradation of SAMHD1, resulting in an increased pool ofcytoplasmic dNTPs available for reverse transcription. In somenon-limiting embodiments, Vpu and Vpx is packaged within the retroviralparticle for any composition or method aspect and embodiment thatincludes a retroviral particle provided herein.

Retroviral particles (e.g. lentiviral particles) included in variousaspects of the present invention are in illustrative embodiments,replication incompetent, especially for safety reasons for embodimentsthat include introducing cells transduced with such retroviral particlesinto a subject. When replication incompetent retroviral particles areused to transduce a cell, retroviral particles are not produced from thetransduced cell. Modifications to the retroviral genome are known in theart to assure that retroviral particles that include the genome arereplication incompetent. However, it will be understood that in someembodiments for any of the aspects provided herein, replicationcompetent recombinant retroviral particles can be used.

A skilled artisan will recognize that the functional elements discussedherein can be delivered to packaging cells and/or to T cells usingdifferent types of vectors, such as expression vectors. Illustrativeaspects of the invention utilize retroviral vectors, and in someparticularly illustrative embodiments lentiviral vectors. Other suitableexpression vectors can be used to achieve certain embodiments herein.Such expression vectors include, but are not limited to, viral vectors(e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus(see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994;Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649,WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655);adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., InvestOpthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al.,Hum Mol Genet 5:591 594, 19%; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40;herpes simplex virus; or a retroviral vector (e.g., Murine LeukemiaVirus, spleen necrosis virus, and vectors derived from retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus), for example a gamma retrovirus; or human immunodeficiencyvirus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi etal., J Virol 73:7812 7816, 1999); and the like.

As disclosed herein, replication incompetent recombinant retroviralparticles are a common tool for gene delivery (Miller, Nature (1992)357:455-460). The ability of replication incompetent recombinantretroviral particles to deliver an unrearranged nucleic acid sequenceinto a broad range of rodent primate and human somatic cells makesreplication incompetent recombinant retroviral particles well suited fortransferring genes to a cell. In some embodiments, the replicationincompetent recombinant retroviral particles can be derived from theAlpharetrovirus genus, the Betaretrovirus genus, the Gammaretrovirusgenus, the Deltaretrovirus genus, the Epsilonretrovirus genus, theLentivirus genus, or the Spumavirus genus. There are many retrovirusessuitable for use in the methods disclosed herein. For example, murineleukemia virus (MLV), human immunodeficiency virus (HTV), equineinfectious anaemia virus (EIAV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV) can be used. A detailed list ofretroviruses may be found in Coffin et al (“Retroviruses” 1997 ColdSpring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 758-763). Details on the genomic structure of some retroviruses maybe found in the art. By way of example, details on HIV may be found fromthe NCBI Genbank (i.e. Genome Accession No. AF033819).

In illustrative embodiments, the replication incompetent recombinantretroviral particles can be derived from the Lentivirus genus. In someembodiments, the replication incompetent recombinant retroviralparticles can be derived from HIV, SIV, or FIV. In further illustrativeembodiments, the replication incompetent recombinant retroviralparticles can be derived from the human immunodeficiency virus (HTV) intire Lentivirus genus. Lentiviruses are complex retroviruses which, inaddition to the common retroviral genes gag, pol and env, contain othergenes with regulatory or structural function. The higher complexityenables the lentivirus to modulate the life cycle thereof, as in thecourse of latent infection. A typical lentivirus is the humanimmunodeficiency virus (HTV), the etiologic agent of AIDS. In vivo, HTVcan infect terminally differentiated cells that rarely divide, such aslymphocytes and macrophages.

In illustrative embodiments, replication incompetent recombinantretroviral particles provided herein contain Vpx polypeptide.

In some embodiments, replication incompetent recombinant retroviralparticles provided herein comprise and/or contain Vpu polypeptide.

In illustrative embodiments, a retroviral particle is a lentiviralparticle. Such retroviral particle typically includes a retroviralgenome within a capsid which is located within a viral envelope.

In some embodiments, DNA-containing viral particles are utilized insteadof recombinant retroviral particles. Such viral particles can beadenoviruses, adeno-associated viruses, herpesviruses,cytomegaloviruses, poxviruses, avipox viruses, influenza viruses,vesicular stomatitis virus (VSV), or Sindbis virus. A skilled artisanwill appreciate how to modify the methods disclosed herein for use withdifferent viruses and retroviruses, or retroviral particles. Where viralparticles are used that include a DNA genome, a skilled artisan willappreciate that functional units can be included in such genomes toinduce integration of all or a portion of the DNA genome of the viralparticle into the genome of a T cell transduced with such virus.

In some embodiments, the HIV RREs and the polynucleotide region encodingHIV Rev can be replaced with N-terminal RGG box RNA binding motifs and apolynucleotide region encoding ICP27. In some embodiments, thepolynucleotide region encoding HIV Rev can be replaced with one or morepolynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.

Provided herein in one aspect is a container, such as a commercialcontainer or package, or a kit comprising the same, comprising isolatedreplication incompetent recombinant retroviral particles according toany of the replication incompetent recombinant retroviral particleaspects provided herein. Furthermore, provided herein in another aspectis a container, such as a commercial container or package, or a kitcomprising the same, comprising isolated packaging cells, inillustrative embodiments isolated packaging cells from a packaging cellline, according to any of the packaging cell and/or packaging cell lineaspects provided herein. In some embodiments, the kit includesadditional containers that include additional reagents such as buffersor reagents used in methods provided herein. Furthermore provided hereinin certain aspects are use of any replication incompetent recombinantretroviral particle provided herein in any aspect, in the manufacture ofa kit for genetically modifying a T cell or NK cell according to anyaspect provided herein. Furthermore provided herein in certain aspectsare use of any packaging cell or packaging cell line provided herein inany aspect, in the manufacture of a kit for producing the replicationincompetent recombinant retroviral particles according to any aspectprovided herein.

Provided herein in one aspect is a commercial container containing areplication incompetent recombinant retroviral particle and instructionsfor the use thereof to treat tumor growth in a subject, wherein thereplication incompetent recombinant retroviral particle comprises in itsgenome a polynucleotide comprising one or more nucleic acid sequencesoperatively linked to a promoter active in T cells and/or NK cells. Insome embodiments, a nucleic acid sequence of the one or more nucleicacid sequences can encode a chimeric antigen receptor (CAR) comprisingan antigen-specific targeting region (ASTR), a transmembrane domain, andan intracellular activating domain. In some embodiments, a nucleic acidsequence of the one or more nucleic acid sequences can encode two ormore inhibitory RN A molecules directed against one or more RNA targets.

The container that contains the recombinant retroviral particles can bea tube, vial, well of a plate, or other vessel for storage of arecombinant retroviral particle. The kit can include two or morecontainers wherein a second or other container can include, for example,a solution or media for transduction of T cells and/or NK cells, and/orthe second or other container can include a pH-modulating pharmacologicagent. Any of these containers can be of industrial strength and grade.The replication incompetent recombinant retroviral particle in suchaspects that include a kit and a nucleic acid encoding an inhibitory RNAmolecule, can be any of the embodiments for such replication incompetentrecombinant retroviral particles provided herein, which include any ofthe embodiments for inhibitory RNA provided herein.

In another aspect, provided herein is the use of a replicationincompetent recombinant retroviral particle in the manufacture of a kitfor genetically modifying a T cell or NK cell, wherein the use of thekit includes: contacting the T cell or NK cell ex vivo with thereplication incompetent recombinant retroviral particle, wherein thereplication incompetent recombinant retroviral particle includes apseudotyping element on a surface and a T cell activation element on thesurface, wherein said contacting facilitates transduction of the T cellor NK cell by the replication incompetent recombinant retroviralparticle, thereby producing a genetically modified T cell or NK cell. Insome embodiments, the T cell or NK cell can be from a subject. In someembodiments, the T cell activation element can be membrane-bound. Insome embodiments, the contacting can be performed for between 1, 2, 3,4, 5, 6, 7, or 8 hours on the low end of the range and 4, 5, 6, 7, 8,10, 12, 15, 18, 21, and 24 hours on the high end of the range, forexample, between 1 and 12 hours. The replication incompetent recombinantretroviral particle for use in the manufacture of a kit can include anyof the aspects, embodiments, or subembodiments discussed elsewhereherein.

In another aspect, provided herein is a pharmaceutical composition fortreating or preventing cancer or tumor growth comprising a replicationincompetent recombinant retroviral particle as an active ingredient. Inanother aspect, provided herein is an infusion composition or otherdelivery solution for treating or preventing cancer or tumor growthcomprising a replication incompetent recombinant retroviral particle.The replication incompetent recombinant retroviral particle of thepharmaceutical composition or infusion composition can include any ofthe aspects, embodiments, or subembodiments discussed above or elsewhereherein.

Genetically Modified T Cells and NK Cells

In embodiments of the methods and compositions herein, geneticallymodified lymphocytes are produced, which themselves are a separateaspect of the invention. Such genetically modified lymphocytes can begenetically modified and/or transduced lymphocytes. In one aspect,provided herein a genetically modified T cell or NK cell is made using amethod according to any aspect for genetically modifying T cells and/orNK cells in blood or a component thereof, provided herein. For example,in some embodiments, the T cell or NK cell has been genetically modifiedto express a first engineered signaling polypeptide. In illustrativeembodiments, the first engineered signaling polypeptide can be alymphoproliferative element or a CAR that includes an antigen-specifictargeting region (ASTR), a transmembrane domain, and an intracellularactivating domain. In some embodiments, the T cell or NK cell canfurther include a second engineered signaling polypeptide that can be aCAR or a lymphoproliferative element. In some embodiments, thelymphoproliferative element can be a chimeric lymphoproliferativeelement. In some embodiments, the T cell or NK cell can further includea pseudotyping element on a surface. In some embodiments, the T cell orNK cell can further include an activation element on a surface. The CAR,lymphoproliferative element, pseudotyping element, and activationelement of the genetically modified T cell or NK cell can include any ofthe aspects, embodiments, or subembodiments disclosed herein. Inillustrative embodiments, the activation element can be anti-CD3antibody, such as an anti-CD3 scFvFc.

In some embodiments, genetically modified lymphocytes are lymphocytessuch as T cells or NK cells that have been genetically modified toexpress a first engineered signaling polypeptide comprising at least onelymphoproliferative element and/or a second engineered signalingpolypeptide comprising a chimeric antigen receptor, which includes anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain. In some embodiments of any of theaspects herein, the NK cells are NKT cells. NKT cells are a subset of Tcells that express CD3 and typically coexpress an op T-cell receptor,but also express a variety of molecular markers that are typicallyassociated with NK cells (such as NK1.1 or CD56).

Genetically modified lymphocytes of the present disclosure possess aheterologous nucleic acid sequence that has been introduced into thelymphocyte by a recombinant DNA method. For example, the heterologoussequence in illustrative embodiments is inserted into the lymphocyteduring a method for transducing the lymphocyte provided herein. Theheterologous nucleic acid is found within the lymphocyte and in someembodiments is or is not integrated into the genome of the geneticallymodified lymphocyte.

In illustrative embodiments, the heterologous nucleic acid is integratedinto the genome of the genetically modified lymphocyte. Such lymphocytesare produced, in illustrative embodiments, using a method fortransducing lymphocytes provided herein, that utilizes a recombinantretroviral particle. Such recombinant retroviral particle can include apolynucleotide that encodes a chimeric antigen receptor that typicallyincludes at least an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain. Providedherein in other sections of this disclosure are various embodiments ofreplication incompetent recombinant retroviral particles andpolynucleotides encoded in a genome of the replication incompetentretroviral particle, that can be used to produce genetically modifiedlymphocytes that themselves form another aspect of the presentdisclosure.

Genetically modified lymphocytes of the present disclosure can beisolated outside the body. For example, such lymphocytes can be found inmedia and other solutions that are used for ex vivo transduction asprovided herein. The lymphocytes can be present in a geneticallyunmodified form in blood that is collected from a subject in methodsprovided herein, and then genetically modified during method oftransduction. The genetically modified lymphocytes can be found inside asubject after they are introduced or reintroduced into the subject afterthey have been genetically modified. The genetically modifiedlymphocytes can be a resting T cell or a resting NK cell, or thegenetically modified T cell or NK cell can be actively dividing,especially after it expresses some of the functional elements providedin nucleic acids that are inserted into the T cell or NK cell aftertransduction as disclosed herein.

Provided herein in one aspect is a transduced and/or geneticallymodified T cell or NK cell, comprising a recombinant polynucleotidecomprising one or more transcriptional units operatively linked to apromoter active in T cells and/or NK cells, in its genome.

In some embodiments, provided herein are genetically modifiedlymphocytes, in illustrative embodiments T cells and/or NK cells, thatrelate to either aspects for transduction of T cells and/or NK cells inblood or a component thereof, that include transcription units thatencode one, two, or more (e.g. 1-10, 2-10, 4-10, 1-6, 2-6, 3-6, 4-6,1-4, 2-4, 3-4) inhibitory RNA molecules. In some embodiments, suchinhibitory RNA molecules are lymphoproliferative elements and therefore,can be included in any aspect or embodiment disclosed herein as thelymphoproliferative element as long as they induce proliferation of a Tcell and/or an NK cell, or otherwise meet a test for alymphoproliferative element provided herein.

Inhibitory RNA molecules directed against a variety of target RNAs canbe used in embodiments of any of the aspects provided herein. Forexample, one, most or all of the one (e.g. two) or more inhibitory RNAmolecules decrease expression of an endogenous TCR. In some embodiments,the RNA target is mRNA transcribed from a gene selected from the groupconsisting of: PD-1, CTLA4, TCR alpha, TCR beta, CD3 zeta, SOCS, SMAD2,a miR-155 target IFN gamma, cCBL, TRAIL2, PP2A, and ABCG1. In someembodiments of this aspect at least one of the one (e.g. two) or moreinhibitory RNA molecules is miR-155.

In some embodiments of the aspect immediately above where the T cell orNK cell comprises one or more (e.g. two or more) inhibitory RNAmolecules and the CAR, or nucleic acids encoding the same, the ASTR ofthe CAR is an MRB ASTR and/or the ASTR of the CAR binds to a tumorassociated antigen. Furthermore, in some embodiments of the aboveaspect, the first nucleic acid sequence is operably linked to ariboswitch, which for example is capable of binding a nucleoside analog,and in illustrative embodiments is an antiviral drug such as acyclovir.

In the methods and compositions disclosed herein, expression ofengineered signaling polypeptides is regulated by a control element, andin some embodiments, the control element is a polynucleotide comprisinga riboswitch. In certain embodiments, the riboswitch is capable ofbinding a nucleoside analog and when the nucleoside analog is present,one or both of the engineered signaling polypeptides are expressed.

Nucleic Acids

The present disclosure provides nucleic acid encoding polypeptides ofthe present disclosure. A nucleic acid will in some embodiments be DNA,including, e.g., a recombinant expression vector. A nucleic acid will insome embodiments be RNA, e.g., in vitro synthesized RNA.

In some embodiments, a nucleic acid provides for production of apolypeptide of the present disclosure, e.g., in a mammalian cell. Inother cases, a subject nucleic acid provides for amplification of thenucleic acid encoding a polypeptide of the present disclosure.

A nucleotide sequence encoding a polypeptide of the present disclosurecan be operably linked to a transcriptional control element, e.g., apromoter, and enhancer, etc.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

Suitable reversible promoters, including reversible inducible promotersare known in the art. Such reversible promoters may be isolated andderived from many organisms, e.g., eukaryotes and prokaryotes.Modification of reversible promoters derived from a first organism foruse in a second organism, e.g., a first prokaryote and a second aeukaryote, a first eukaryote and a second a prokaryote, etc., is wellknown in the art. Such reversible promoters, and systems based on suchreversible promoters but also comprising additional control proteins,include, but are not limited to, alcohol regulated promoters (e.g.,alcohol dehydrogenase I (alcA) gene promoter, promoters responsive toalcohol transactivator proteins (AlcR), etc.), tetracycline regulatedpromoters, (e.g., promoter systems including TetActivators, TetON,TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoidreceptor promoter systems, human estrogen receptor promoter systems,retinoid promoter systems, thyroid promoter systems, ecdysone promotersystems, mifepristone promoter systems, etc.), metal regulated promoters(e.g., metallothionein promoter systems, etc.), pathogenesis-relatedregulated promoters (e.g., salicylic acid regulated promoters, ethyleneregulated promoters, benzothiadiazole regulated promoters, etc.),temperature regulated promoters (e.g., heat shock inducible promoters(e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), lightregulated promoters, synthetic inducible promoters, and the like.

In some instances, the locus or construct or trails gene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99, the disclosure of whichis incorporated herein by reference). Any suitable combination ofrecombinase, endonuclease, ligase, recombination sites, etc. known tothe art may be used in generating an irreversibly switchable promoter.Methods, mechanisms, and requirements for performing site-specificrecombination, described elsewhere herein, find use in generatingirreversibly switched promoters and are well known in the art, see,e.g., Grindley et al. (2006) Annual Review of Biochemistry, 567-605 andTropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury,Mass.), the disclosures of which are incorporated herein by reference.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an Neri (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood117:1565.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADHl promoter, a PGKlpromoter, an ENO promoter, a PYKl promoter and the like; or aregulatable promoter such as a GALI promoter, a GALlO promoter, an ADH2promoter, a PH05 promoter, a CUPl promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYCl promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADCl promoter, a TRPlpromoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TPlpromoter, and AOXl (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a tippromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a tip/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacterial., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mal. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spv promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mal. Microbial. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and PLambda. Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (Lacirepressor protein changes conformation when contacted with lactose,thereby preventing the Laci repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a polypeptide of the disclosure can bepresent in an expression vector and/or a cloning vector. Nucleotidesequences encoding two separate polypeptides can be cloned in the sameor separate vectors. An expression vector can include a selectablemarker, an origin of replication, and other features that provide forreplication and/or maintenance of the vector. Suitable expressionvectors include, e.g., plasmids, viral vectors, and the like.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating subjectrecombinant constructs. The following bacterial vectors are provided byway of example: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). The following eukaryotic vectors are provided by way ofexample: pWLneo, pSV2cat, pOG44, PXRl, pSG (Stratagene) pSVK3, pBPV,pMSG, and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present.

As noted above, in some embodiments, a nucleic acid encoding apolypeptide of the present disclosure will in some embodiments be RNA,e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNAare known in the art; any known method can be used to synthesize RNAincluding a nucleotide sequence encoding a polypeptide of the presentdisclosure. Methods for introducing RNA into a host cell are known inthe art. See, e.g., Zhao et al. (2010) Cancer Res. 15:9053. IntroducingRNA including a nucleotide sequence encoding a polypeptide of thepresent disclosure into a host cell can be carried out in vitro or exvivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxicT lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNAcomprising a nucleotide sequence encoding a polypeptide of the presentdisclosure.

Various aspects and embodiments that include a polynucleotide, a nucleicacid sequence, and/or a transcriptional unit, and/or a vector includingthe same, further include one or more of a Kozak-type sequence (alsocalled a Kozak-related sequence herein), a woodchuck hepatitis viruspost-transcriptional regulatory element (WPRE), and a double stop codonor a triple stop codon, wherein one or more stop codons of the doublestop codon or the triple stop codon define a termination of a readingfrom of at least one of the one or more transcriptional units. Incertain embodiments, a polynucleotide a nucleic acid sequence, and/or atranscriptional unit, and/or a vector including the same, furtherincludes a Kozak-type sequence having a 5′ nucleotide within 10nucleotides upstream of a start codon of at least one of the one or moretranscriptional units. Kozak determined the Kozak consensus sequence,(GCC)GCCRCCATG (SEQ ID NO:327), for 699 vertebrate mRNAs, where R is apurine (A or G) (Kozak. Nucleic Acids Res. 1987 Oct. 26;15(20):8125-48). In one embodiment the Kozak-type sequence is orincludes CCACCAT/UG(G) (SEQ ID NO:328), CCGCCAT/UG(G) (SEQ ID NO:329),GCCGCCGCCAT/UG(G) (SEQ ID NO:330), or GCCGCCACCAT/UG(G) (SEQ ID NO:331)(with nucleotides in parenthesis representing optional nucleotides andnucleotides separated by a slash indicated different possiblenucleotides at that position, for example depending on whether thenucleic acid is DNA or RNA. In these embodiments that include the AU/TGstart codon, the A can be considered position 0. In certain illustrativeembodiments, the nucleotides at −3 and +4 are identical, for example the−3 and +4 nucleotides can be G. In another embodiment the Kozak-typesequence includes an A or G in the 3rd position upstream of ATG whereATG is the start codon. In another embodiment the Kozak-type sequenceincludes an A or G in the 3rd position upstream of AUG where AUG is thestart codon. In an illustrative embodiment, the Kozak sequence is(GCC)GCCRCCATG (SEQ ID NO:327), where R is a purine (A or G). In anillustrative embodiment, the Kozak-type sequence is GCCGCCACCAUG (SEQ IDNO:332). In another embodiment, which can be combined with the precedingembodiment that includes a Kozak-type sequence and/or the followingembodiment that includes triple stop codon, the polynucleotide includesa WPRE element. WPREs have been characterized in the art (See e.g.,(Higashimoto et al., Gene Ther. 2007; 14: 1298)) and as illustrated inWO2019/055946. In some embodiments, the WPRE element is located 3′ of astop codon of the one or more transcriptional units and 5′ to a 3′ LTRof the polynucleotide. In another embodiment, which can be combined witheither or both of the preceding embodiments (i.e. an embodiment whereinthe polynucleotide includes a Kozak-type sequence and/or an embodimentwherein the polynucleotide includes a WPRE), the one or moretranscriptional units terminates with one or more stop codons of adouble stop codon or a triple stop codon, wherein the double stop codonincludes a first stop codon in a first reading frame and a second stopcodon in a second reading frame, or a first stop codon in frame with asecond stop codon, and wherein the triple stop codon includes a firststop codon in a first reading frame, a second stop codon in a secondreading frame, and a third stop codon in a third reading frame, or afirst stop codon in frame with a second stop codon and a third stopcodon.

A triple stop codon herein includes three stop codons, one in eachreading frame, within 10 nucleotides of each other, and preferablyhaving overlapping sequence, or three stop codons in the same readingframe, preferably at consecutive codons. A double stop codon means twostop codons, each in a different reading frame, within 10 nucleotides ofeach other, and preferably having overlapping sequences, or two stopcodons in the same reading frame, preferably at consecutive codons.

In some of the methods and compositions disclosed herein, theintroduction of DNA into PBMCs, B cells, T cells and/or NK cells andoptionally the incorporation of the DNA into the host cell genome, isperformed using methods that do not utilize replication incompetentrecombinant retroviral particles. For example, other viral vectors canbe utilized, such as those derived from adenovirus, adeno-associatedvirus, or herpes simplex virus-1, as non-limiting examples.

In some embodiments, methods provided herein can include transfectingtarget cells with non-viral vectors. In any of the embodiments disclosedherein that utilize non-viral vectors to transfect target cells, thenon-viral vectors, including naked DNA, can be introduced into thetarget cells, such as for example, PBMCs, B cells, T cells and/or NKcells using methods that include electroporation, nucleofection,liposomal formulations, lipids, dendrimers, cationic polymers such aspoly(ethylenimine) (PEI) and poly(l-lysine) (PLL), nanoparticles,cell-penetrating peptides, microinjection, and/or non-integratinglentiviral vectors. In some embodiments. DNA can be introduced intotarget cells, such as PBMCs, B cells, T cells and/or NK cells in acomplex with liposomes and protamine. Other methods for transfecting Tcells and/or NK cells ex vivo that can be used in embodiments of methodsprovided herein, are known in the art (see e.g., Morgan and Boyerinas,Biomedicines. 2016 Apr. 20; 4(2). pii: E9, incorporated by referenceherein in its entirety).

In some embodiments of method provided herein. DNA can be integratedinto the genome using transposon-based carrier systems byco-transfection, co-nucleofection or co-electroporation of target DNA asplasmid containing the transposon ITR fragments in 5′ and 3′ ends of thegene of interest and transposase carrier system as DNA or mRNA orprotein or site specific serine recombinases such as phiC31 thatintegrates the gene of interest in pseudo attP sites in the humangenome, in this instance the DNA vector contains a 34 to 40 bp attB sitethat is the recognition sequence for the recombinase enzyme (BhaskarThyagarajan et al. Site-Specific Genomic Integration in Mammalian CellsMediated by Phage φC31 Integrase, Mol Cell Biol. 2001 June; 21(12):3926-3934) and co transfected with the recombinase. For T cells and/orNK cells, transposon-based systems that can be used in certain methodsprovided herein utilize the Sleeping Beauty DNA carrier system (seee.g., U.S. Pat. No. 6,489,458 and U.S. patent application Ser. No.15/434,595, incorporated by reference herein in their entireties), thePiggyBac DNA carrier system (see e.g., Manuri et al., Hum Gene Ther.2010 April; 21(4):427-37, incorporated by reference herein in itsentirety), or the Tol2 transposon system (see e.g., Tsukahara et al.,Gene Ther. 2015 February; 22(2): 209-215, incorporated by referenceherein in its entirety) in DNA, mRNA, or protein form. In someembodiments, the transposon and/or transposase of the transposon-basedvector systems can be produced as a minicircle DNA vector beforeintroduction into T cells and/or NK cells (see e.g., Hudecek et al.,Recent Results Cancer Res. 2016; 209:37-50 and Monjezi et al., Leukemia.2017 January; 31(1):186-194, incorporated by reference herein in theirentireties). The CAR or lymphoproliferative element can also beintegrated into the defined and specific sites in the genome usingCRISPR or TALEN mediated integration, by adding 50-1000 bp homology armshomologous to the integration 5′ and 3′ of the target site (Jae SeongLee et al. Scientific Reports 5, Article number: 8572 (2015),Site-specific integration in CHO cells mediated by CRISPR/Cas9 andhomology-directed DNA repair pathway). CRISPR or TALEN providespecificity and genomic-targeted cleavage and the construct will beintegrated via homology-mediated end joining (Yao X at al. Cell Res.2017 June; 27(6):801-814. doi: 10.1038/cr.2017.76. Epub 2017 May 19).The CRISPR or TALEN can be co-transfected with target plasmid as DNA,mRNA, or protein.

Inhibitory RNA Molecules

Embodiments of any of the aspects provided herein can includerecombinant retroviral particles whose genomes are constructed to induceexpression of one or more, and in illustrative embodiments two or more,inhibitory RNA molecules, such as for example, a miRNA or shRNA, afterintegration into a host cell, such as a lymphocyte (e.g. a T cell and/oran NK cell). Such inhibitory RNA molecules can be encoded withinintrons, including for example, an EF1-a intron. This takes advantage ofthe present teachings of methods to maximize the functional elementsthat can be included in a packageable retroviral genome to overcomeshortcomings of prior teachings and maximize the effectiveness of suchrecombinant retroviral particles in adoptive T cell therapy.

In some embodiments, the inhibitory RNA molecule includes a 5′ strandand a 3′ strand (in some examples, sense strand and antisense strand)that are partially or fully complementary to one another such that thetwo strands are capable of forming a 18-25 nucleotide RNA duplex withina cellular environment. The 5′ strand can be 18, 19, 20, 21, 22, 23, 24,or 25 nucleotides in length, and the 3′ strand can be 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length. The 5′ strand and the 3′ strandcan be the same or different lengths, and the RNA duplex can include oneor more mismatches. Alternatively, the RNA duplex has no mismatches.

The inhibitory RNA molecules included in compositions and methodsprovided herein, in certain illustrative examples, do not exist and/orare not expressed naturally in T cells into whose genome they areinserted. In some embodiments, the inhibitory RNA molecule is a miRNA oran shRNA. In some embodiments, where reference is made herein or inpriority filings, to a nucleic acid encoding an siRNA, especially in acontext where the nucleic acid is part of a genome, it will beunderstood that such nucleic acid is capable of forming an siRNAprecursor such as miRNA or shRNA in a cell that is processed by DICER toform a double stranded RNA that typically interacts with, or becomespart of a RISK complex. In some embodiments, an inhibitory molecule ofan embodiment of the present disclosure is a precursor of a miRNA, suchas for example, a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA.In some embodiments, the miRNA or shRNA are artificially derived (i.e.artificial miRNAs or siRNAs). In other embodiments, the inhibitory RNAmolecule is a dsRNA (either transcribed or artificially introduced) thatis processed into an siRNA or the siRNA itself. In some embodiments, themiRNA or shRNA has a sequence that is not found in nature, or has atleast one functional segment that is not found in nature, or has acombination of functional segments that are not found in nature.

In some embodiments, inhibitory RNA molecules are positioned in thefirst nucleic acid molecule in a series or multiplex arrangement suchthat multiple miRNA sequences are simultaneously expressed from a singlepolycistronic miRNA transcript. In some embodiments, the inhibitory RNAmolecules can be adjoined to one another either directly or indirectlyby non-functional linker sequence(s). The linker sequence in someembodiments, is between 5 and 120 nucleotides in length, and in someembodiments can be between 10 and 40 nucleotides in length, asnon-limiting examples. In illustrative embodiments the first nucleicacid sequence encoding one or more (e.g. two or more) inhibitory RNAsand the second nucleic acid sequence encoding a CAR (e.g. an MRB-CAR)are operably linked to a promoter that is active constitutively or thatcan be induced in a T cell or NK cell. As such, the inhibitory RNAmolecule(s) (e.g. miRNAs) as well as the CAR are expressed in apolycistronic manner. Additionally, functional sequences can beexpressed from the same transcript. For example, any of thelymphoproliferative elements provided herein that are not inhibitory RNAmolecules, can be expressed from the same transcript as the CAR and theone or more (e.g. two or more) inhibitory RNA molecules.

In some embodiments, the inhibitory RNA molecule is a naturallyoccurring miRNA such as but not limited to miR-155. Alternatively,artificial miRNAs can be produced in which sequences capable of forminga hybridizing/complementary stem structure and directed against a targetRNA, are placed in a miRNA framework that includes microRNA flankingsequences for microRNA processing and a loop, which can optionally bederived from the same naturally occurring miRNA as the flankingsequences, between the stem sequences. Thus, in some embodiments, aninhibitory RNA molecule includes from 5′ to 3′ orientation: a 5′microRNA flanking sequence, a 5′ stem, a loop, a 3′ stem that ispartially or fully complementary to said 5′ stem, and a 3′ microRNAflanking sequence. In some embodiments, the 5′ stem (also called a 5′arm herein) is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.In some embodiments, the 3′ stem (also called a 3′ arm herein) is 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In someembodiments, the loop is 3 to 40, 10 to 40, 20 to 40, or 20 to 30nucleotides in length, and in illustrative embodiments the loop can be18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, onestem is two nucleotides longer than the other stem. The longer stem canbe the 5′ or the 3′ stem.

In some embodiments, the 5′ microRNA flanking sequence, 3′ microRNAflanking sequence, or both, are derived from a naturally occurringmiRNA, such as but not limited to miR-155, miR-30, miR-17-92, miR-122,and miR-21. In certain embodiments, the 5′ microRNA flanking sequence,3′ microRNA flanking sequence, or both, are derived from a miR-155, suchas, e.g., the miR-155 from Mus musculus or Homo sapiens. Inserting asynthetic miRNA stem-loop into a miR-155 framework (i.e. the 5′ microRNAflanking sequence, the 3′ microRNA flanking sequence, and the loopbetween the miRNA 5′ and 3′ stems) is known to one of ordinary skill inthe art (Chung, K. et al. 2006. Nucleic Acids Research. 34(7):e53; U.S.Pat. No. 7,387,896). The SIBR (synthetic inhibitory BIC-derived RNA)sequence (Chung et al. 2006 supra), for example, has a 5′ microRNAflanking sequence consisting of nucleotides 134-161 (SEQ ID NO:333) ofthe Mus musculus BIC noncoding mRNA (Genbank ID AY096003.1) and a 3′microRNA flanking sequence consisting of nucleotides 223-283 of the Musmusculus BIC noncoding mRNA (Genbank ID AY096003.1). In one study, theSIBR sequence was modified (eSIBR) to enhance expression of miRNAs(Fowler, D. K. et al. 2015. Nucleic acids Research 44(5):e48). In someembodiments of the present disclosure, miRNAs can be placed in the SIBRor eSIBR miR-155 framework. In illustrative embodiments herein, miRNAsare placed in a miR-155 framework that includes tire 5′ microRNAflanking sequence of miR-155 represented by SEQ ID NO:333, the 3′microRNA flanking sequence represented by SEQ ID NO:334 (nucleotides221-265 of the Mus musculus BIC noncoding mRNA); and a modified miR-155loop (SEQ ID NO:335). Thus, in some embodiments, the 5′ microRNAflanking sequence of miR-155 is SEQ ID NO:333 or a functional variantthereof, such as, for example, a sequence that is the same length as SEQID NO:333, or 95%, 90%, 85%, 80%75% or 50% as long as SEQ ID NO: 333 oris 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides orless, 85 nucleotides or less. 80 nucleotides or less, 75 nucleotides orless, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides orless, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides orless, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides orless, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:333. In someembodiments, the 3′ microRNA flanking sequence of miR-155 is SEQ IDNO:334 or a functional variant thereof, such as, for example, the samelength as SEQ ID NO:334, or 95%, 90%, 85% 80%75%, or 50% as long as SEQID NO:334 or is a sequence that is 100 nucleotides or less, 95nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less;and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%identical to SEQ ID NO:334. However, any known microRNA framework thatis functional to provide proper processing within a cell of miRNAsinserted therein to form mature miRNA capable of inhibiting expressionof a target mRNA to which they bind, is contemplated within the presentdisclosure.

In some embodiments, at least one, at least two, at least three, or atleast four of the inhibitory RNA molecules encoded by a nucleic acidsequence in a polynucleotide of a replication incompetent recombinantretroviral particle has the following arrangement in the 5′ to 3′orientation: a 5′ microRNA flanking sequence, a 5′ stem, a loop, a 3′stem that is partially or folly complementary to said 5′ stem, and a 3′microRNA flanking sequence. In some embodiments, all of the inhibitoryRNA molecules have the following arrangement in the 5′ to 3′orientation: a 5′ microRNA flanking sequence, a 5′ stem, a loop, a 3′stem that is partially or folly complementary to said 5′ stem, and a 3′microRNA flanking sequence. As disclosed herein, the inhibitory RNAmolecules can be separated by one or more linker sequences, which insome embodiments have no function except to act as spacers betweeninhibitory RNA molecules.

In some embodiments, where two or more inhibitory RNA molecules (in someexamples, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 inhibitory RNA molecules) areincluded, these inhibitory RNA molecules are directed against the sameor different RNA targets (such as e.g. mRNAs transcribed from genes ofinterest). In illustrative embodiments, between 2 and 10, 2 and 8, 2 and6, 2 and 5, 3 and 5, 3 and 6, or 4 inhibitory RNA molecules are includedin the first nucleic acid sequence. In an illustrative embodiment, fourinhibitory RNA molecules are included in the first nucleic acidsequence.

In some embodiments, the one or more inhibitor RNA molecules are one ormore lymphoproliferative elements, accordingly, in any aspect orembodiment provided herein that includes a lymphoproliferative element,unless incompatible therewith (e.g. a polypeptide lymphoproliferativeelement), or already state therein. In some embodiments, the RNA targetsare mRNAs transcribed from genes that are expressed by T cells such asbut not limited to PD-1 (prevent inactivation); CTLA4 (preventinactivation); TCRα (safety—prevent autoimmunity); TCRb (safety—preventautoimmunity); CD3Z (safety—prevent autoimmunity); SOCS1 (preventinactivation); SMAD2 (prevent inactivation); a miR-155 target (promoteactivation); IFN gamma (reduce CRS); cCBL (prolong signaling); TRAIL2(prevent death); PP2A (prolong signaling); ABCG1 (increase cholesterolmicrodomain content by limiting clearance of cholesterol). Inillustrative examples, miRNAs inserted into the genome of T cells inmethods provided herein, are directed at targets such that proliferationof the T cells is induced and/or enhanced and/or apoptosis issuppressed.

In some embodiments, the RNA targets include mRNAs that encodecomponents of the T cell receptor (TCR) complex. Such components caninclude components for generation and/or formation of a T cell receptorcomplex and/or components for proper functioning of a T cell receptorcomplex. Accordingly, in one embodiment at least one of tire two or moreof inhibitory RNA molecules causes a decrease in the formation and/orfunction of a TCR complex, in illustrative embodiments one or moreendogenous TCR complexes of a T cell. The T cell receptor complexincludes TCRa, TCRb, CD3d, CD3e, CD3g, and CD3z. It is known that thereis a complex interdependency of these components such that a decrease inthe expression of any one subunit will result in a decrease in theexpression and function of the complex. Accordingly, in one embodimentthe RNA target is an mRNA expressing one or more of TCRa, TCRb, CD3d,CD3e, CD3g, and CD3z endogenous to a transduced T cell. In certainembodiments, the RNA target is mRNA transcribed from the endogenous TCRαor TCRβ gene of the T cell whose genome comprises the first nucleic acidsequence encoding the one or more miRNAs. In illustrative embodiments,the RNA target is mRNA transcribed from the TCRα gene. In certainembodiments, inhibitory RNA molecules directed against mRNAs transcribedfrom target genes with similar expected utilities can be combined. Inother embodiments, inhibitory RNA molecules directed against targetmRNAs transcribed from target genes with complementary utilities can becombined. In some embodiments, the two or more inhibitory RNA moleculesare directed against the mRNAs transcribed from the target genes CD3Z,PD1, SOCS1, and/or IFN gamma.

In some embodiments, the inhibitory RNA, for example miRNA, targets mRNAencoding Cbl Proto-Oncogene (RNF55) (also known as cCBL and RNF55)(HGNC: 1541, Entrez Gene: 867, OMIM: 165360), T-Cell Receptor T3 ZetaChain (CD3z) (HGNC: 1677, Entrez Gene: 919, OMIM: 186780), PD1, CTLA4, TCell Immunoglobulin Mucin 3 (TIM3) (also known as Hepatitis A VirusCellular Receptor 2) (HGNC: 18437 Entrez Gene: 84868, OMIM: 606652),Lymphocyte Activating 3 (LAG3) (HGNC: 6476, Entrez Gene: 3902, OMIM:153337), SMAD2, TNF Receptor Superfamily Member 10b (TNFRSF10B) (HGNC:11905, Entrez Gene: 8795, OMIM: 603612), Protein Phosphatase 2 CatalyticSubunit Alpha (PPP2CA) (HGNC: 9299, Entrez Gene: 5515, OMIM: 176915),Tumor Necrosis Factor Receptor Superfamily Member 6 (TNFRSF6) (alsoknown as Fas Cell Surface Death Receptor (FAS)) (HGNC: 11920, EntrezGene: 355, OMIM: 134637), B And T Lymphocyte Associated (BTLA) (HGNC:21087, Entrez Gene: 151888, OMIM: 607925), T Cell Immunoreceptor With IgAnd ITIM Domains (TIGIT) (HGNC: 26838, Entrez Gene: 201633, OMIM:612859), Adenosine A2a Receptor (ADORA2A or A2AR) (HGNC: 263, EntrezGene: 135, OMIM: 102776), Aryl Hydrocarbon Receptor (AHR) (HGNC: 348,Entrez Gene: 196, OMIM: 600253), Eomesodermin (EOMES) (HGNC: 3372,Entrez Gene: 8320, OMIM: 604615), SMAD Family Member 3 (SMAD3) (HGNC:6769, Entrez Gene: 4088, OMIM: 603109), SMAD Family Member 4 (SMAD4)(GNC: 6770, Entrez Gene: 4089, OMIM: 600993), TGFBR2, ProteinPhosphatase 2 Regulatory Subunit B delta (PPP2R2D) (HGNC: 23732, EntrezGene: 55844, OMIM: 613992), Tumor Necrosis Factor Ligand SuperfamilyMember 6 (TNFSF6) (also known as FASL) (HGNC: 11936, Entrez Gene: 356,OMIM: 134638), Caspase 3 (CASP3) HGNC: 1504, Entrez Gene: 836, OMIM:600636), Suppressor Of Cytokine Signaling 2 (SOCS2) (HGNC: 19382, EntrezGene: 8835, OMIM: 605117), Kruppel Like Factor 10 (KLF10) (also known asTGFB-Inducible Early Growth Response Protein 1 (TIEG1)) (HGNC: 11810,Entrez Gene: 7071, OMIM: 601878), JunB Proto-Oncogene, AP-1Transcription Factor Subunit (JunB) (HGNC: 6205, Entrez Gene: 3726,OMIM: 165161), Cbx3, Tet Methylcytosine Dioxygenase 2 (Tet2) (HGNC:25941, Entrez Gene: 54790, OMIM: 612839), Hexokinase 2 (HK2) (HGNC:4923, Entrez Gene: 3099, OMIM: 601125), Src homology region 2domain-containing phosphatase-1 (SHP1) (HGNC: 9658, Entrez Gene: 5777,OMIM: 176883) Src homology region 2 domain-containing phosphatase-2(SHP2) (HGNC: 9644, Entrez Gene: 5781, OMIM: 176876); or in someembodiments encoding TIM3, LAG3, TNFRSF10B, PPP2CA, TNFRSF6 (FAS), BTLA,TIGIT, A2AR, AHR, EOMES, SMAD3, SMAD4, PPP2R2D, TNFSF6 (FASL), CASP3,SOCS2, TIEG1, JunB, Cbx3, Tet2, HK2, SHP1, or SHP2. In some illustrativeembodiments, the inhibitory RNA, for example miRNA, targets mRNAencoding FAS, AHR, CD3z, cCBL, Chromobox 1 (Cbx) (HGNC: 1551, EntrezGene: 10951, OMIM: 604511), HK2, FASL, SMAD4, or EOMES; or in someillustrative embodiments, the inhibitory RNA, for example miRNA, targetsmRNA encoding FAS, AHR, Cbx3, HK2, FASL, SMAD4, or EOMES; or in someillustrative embodiments, the inhibitory RNA, for example miRNA, targetsmRNA encoding AHR, Cbx3, HK2, SMAD4, or EOMES.

In some further illustrative embodiments, a vector or genome herein,includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 of theinhibitory RNA (e.g. miRNA) identified herein, for example in theparagraph immediately above. In some further illustrative embodiments, avector or genome herein, includes 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3,4, 5, 6, 7, or 8 inhibitory RNA (e.g. miRNA) that target mRNA encodingFAS, cCBL, AHR, CD3z, Cbx, EOMES, or HK2, or a combination of 1 or moreinhibitory RNA that target such mRNA. In some further illustrativeembodiments, a vector or genome herein, includes 2 or more, 2-10, 2-8,2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g. miRNA) that targetmRNA encoding AHR, Cbx3, HOMES, or HK2, or a combination of 1 or moreinhibitory RNA that target such mRNA.

In some embodiments provided herein, the two or more inhibitory RNAmolecules can be delivered in a single intron, such as but not limitedto EF1-a intron A. Intron sequences that can be used to harbor miRNAsfor the present disclosure include any intron that is processed within aT cell. As indicated herein, one advantage of such an arrangement isthat this helps to maximize the ability to include miRNA sequenceswithin the size constraints of a retroviral genome used to deliver suchsequences to a T cell in methods provided herein. This is especiallytrue where an intron of the first nucleic acid sequence includes all ora portion of a promoter sequence used to express that intron, a CARsequence, and other functional sequences provided herein, such aslymphoproliferative element(s) that are not inhibitory RNA molecules, ina polycistronic manner. Sequence requirements for introns are known inthe art. In some embodiments, such intron processing is operably linkedto a riboswitch, such as any riboswitch disclosed herein. Thus, theriboswitch can provide a regulatory element for control of expression oftire one or more miRNA sequences on the first nucleic acid sequence.Accordingly, in illustrative embodiments provided herein is acombination of an miRNA directed against an endogenous T cell receptorsubunit wherein the expression of the miRNA is regulated by ariboswitch, which can be any of the riboswitches discussed herein.

In some embodiments, inhibitory RNA molecules can be provided onmultiple nucleic acid sequences that can be included on the same or adifferent transcriptional unit. For example, a first nucleic acidsequence can encode one or more inhibitory RNA molecules and beexpressed from a first promoter and a second nucleic acid sequence canencode one or more inhibitory RNA molecules and be expressed from asecond promoter. In illustrative embodiments, two or more inhibitory RNAmolecules are located on a first nucleic acid sequence that is expressedfrom a single promoter. The promoter used to express such miRNAs, aretypically promoters that are inactive in a packaging cell used toexpress a retroviral particle that will deliver the miRNAs in its genometo a target T cell, but such promoter is active, either constitutivelyor in an inducible manner, within a T cell. The promoter can be a Pol I,Pol II, or Pol III promoter. In some illustrative embodiments, thepromoter is a Pol II promoter.

Characterization and Commercial Production Methods

The present disclosure provides various methods and compositions thatcan be used as research reagents in scientific experimentation and forcommercial production. Such scientific experimentation can includemethods for characterization of lymphocytes, such as NK cells and inillustrative embodiments, T cells using methods for geneticallymodifying, for example transducing lymphocytes provided herein. Suchmethods for example, can be used to study activation of lymphocytes andthe detailed molecular mechanisms by which activation makes such cellstransducible. Furthermore, provided herein are genetically modifiedlymphocytes that will have utility for example, as research tools tobetter understand factors that influence T cell proliferation andsurvival. Such genetically modified lymphocytes, such as NK cells and inillustrative embodiments T cells, can furthermore be used for commercialproduction, for example for the production of certain factors, such asgrowth factors and immunomodulatory agents, that can be harvested andtested or used in the production of commercial products.

The scientific experiments and/or the characterization of lymphocytescan include any of the aspects, embodiments, or subembodiments providedherein useful for analyzing or comparing lymphocytes. In someembodiments, T cells and/or NK cells can be transduced with thereplication incompetent recombinant retroviral particles provided hereinthat include polynucleotides. In some embodiments, transduction of the Tcells and/or NK cells can include polynucleotides that includepolynucleotides encoding polypeptides of the present disclosure, forexample, CARs, lymphoproliferative elements, and/or activation elements.In some embodiments, the polynucleotides can include inhibitory RNAmolecules as discussed elsewhere herein. In some embodiments, thelymphoproliferative elements can be chimeric lymphoproliferativeelements.

EXEMPLARY EMBODIMENTS

Provided in this Exemplary Embodiments section are exemplary aspects andembodiments provided herein and further discussed throughout thisspecification. For the sake of brevity and convenience, all of thedisclosed aspects and embodiments and all of the possible combinationsof the disclosed aspects and embodiments are not listed in this section.It wilt be understood that embodiments are provided that are specificembodiments for many aspects, as discussed in this entire disclosure. Itis intended in view of the full disclosure herein, that any individualembodiment recited below or in this full disclosure can be combined withany aspect recited below or in this full disclosure where it is anadditional element that can be added to an aspect or because it is anarrower element for an element already present in an aspect. Suchcombinations are discussed more specifically in other sections of thisdetailed description.

Unless incompatible with, or already stated in an aspect or embodiment,for any of the methods for genetically modifying and/or transducinglymphocytes (e.g. PBMCs, or T cells and/or NK cells), or uses thatinclude such methods, or genetically modified cells produced using suchmethods, and any other method or product by process, provided herein,including but not limited to in this Exemplary Embodiments section thatincludes a contacting step of contacting retroviral particles withlymphocytes (e.g. PBMCs, or T cells and/or NK cells), in certainembodiments, the contacting step can be performed (or can occur) forbetween 30 seconds and 72 hours, for example, between 1 minute and 12hours, or between 5 minutes and 12 hours, 10 hours, 8 hours, 6 hours, 4hours, 2 hours, 1 hour, 30 minutes, or 15 minutes. In some embodiments,the contacting can be performed for less than 24 hours, for example,less than 12 hours, less than 8 hours, less than 4 hours, and inillustrative embodiments less than 2 hours, less than 1 hour, less than30 minutes or less than 15 minutes, but in each case there is at leastan initial contacting step in which retroviral particles and cells arebrought into contact in suspension in a transduction reaction mixture.Such suspension can include allowing cells and retroviral particles tosettle or causing such settling through application of a force, such asa centrifugal force, to the bottom of a vessel or chamber. However, incertain illustrative embodiments, such force is less than that used forspinoculation, as discussed in more detail herein. After such initialcontacting, there can be an additional optional incubating in thereaction mixture containing cells and retroviral particles in suspensionin the reaction mixture for the time periods specified without removingretroviral particles that remain free in solution and not associatedwith cells. In illustrative embodiments, the contacting can be performed(or can occur) for between 30 seconds or 1, 2, 5, 10, 15, 30 or 45minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours on the low end of the range,and between 10 minutes, 15 minutes, 30 minutes, or 1, 2, 4, 6, 8, 10,12, 18, 24, 36, 48, and 72 hours on the high end of the range. Incertain illustrative embodiments, the contacting step can be performedfor between 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or30 minutes on the low end of the range and 1 hour, 2 hours, 4 hours, 6hours, 8 hours, 10 hours, or 12 hours on the high end of the range. Insome embodiments, the contacting step is performed for between 30seconds, 1 minute, and 5 minutes on the low end of the range, and 10minutes, 15 minutes, 30 minutes, 45 minutes, or 60 minutes on the highend of the range. In another illustrative embodiment, the contacting isperformed for between an initial contacting step only (without anyfurther incubating in the reaction mixture including the retroviralparticles free in suspension and cells in suspension) without anyfurther incubation in the reaction mixture, or a 5 minute or less, 10minute or less, 15 minute or less, 30 minute or less, or 1 hour or lessincubation in the reaction mixture. In some embodiments, the replicationincompetent recombinant retroviral particles can be immediately washedout after adding them to the cell(s) to be genetically modified and/ortransduced such that the contacting time is carried out for the lengthof time it takes to wash out the replication incompetent recombinantretroviral particles. Accordingly, typically the contacting includes atleast an in initial contacting step in which a retroviral particle(s)and a cell(s) are brought into contact in suspension in a transductionreaction mixture. Such methods can be performed without prioractivation.

In any of the aspects and embodiments provided herein that include, oroptionally include, a nucleic acid sequence encoding an inhibitory RNAmolecule, including, but not limited to, aspects and embodimentsprovided in this Exemplary Embodiments section, unless already statedtherein, or incompatible therewith, such nucleic acid sequence isincluded and such inhibitory RNA molecule, in certain embodiments,targets any of the gene (e.g. mRNAs encoding) targets identified forexample in the Inhibitory RNA Molecules section herein; or in certainembodiments targets TCRa, TCRb, SOCS1, miR155 target, IFN gamma, cCBL,TRAIL2, PP2A, ABCG1, cCBL, CD3z, CD3z, PD1, CTLA4, TIM3, LAG3, SMAD2,TNFRSF10B, PPP2CA, TNFRSF6 (FAS), BTLA, TIGIT, A2AR, AHR, HOMES, SMAD3,SMAD4, TGFBR2, PPP2R2D, TNFSF6 (FASL), CASP3, SOCS2, TIEG1, JunB, Cbx3,Tet2, HK2, SHP1, or SHP2; or in certain embodiments targets cCBL, CD3z,CD3z, PD1, CTLA4, TIM3, LAG3, SMAD2, TNFRSF10B, PPP2CA, TNFRSF6 (FAS),BTLA, TIGIT, A2AR, AHR, HOMES, SMAD3, SMAD4, TGFBR2, PPP2R2D, TNFSF6(FASL), CASP3, SOCS2, TIEG1, JunB, Cbx3, Tet2, HK2, SHP1, or SHP2; or incertain embodiments targets mRNA encoding TIM3, LAG3, TNFRSF10B, PPP2CA,TNFRSF6 (FAS), BTLA, TIGIT, A2AR, AHR, HOMES, SMAD3, SMAD4, PPP2R2D,TNFSF6 (FASL), CASP3, SOCS2, TIEG1, JunB, Cbx3, Tet2, HK2, SHP1, orSHP2; or in certain illustrative embodiments, targets mRNA encoding FAS,AHR, CD3z, cCBL, Cbx, HK2, FASL, SMAD4, or HOMES; or in certainillustrative embodiments targets mRNA encoding FAS, AHR, Cbx3, HK2,FASL, SMAD4, or HOMES; or in further illustrative embodiments targetsmRNA encoding AHR, Cbx3, HK2, SMAD4, or HOMES. In some embodiments, theinhibitory RNA molecule includes at least one of the sequences of SEQ IDNOs:342-449. In some embodiments, the inhibitory RNA molecule includesat least one of the sequences of SEQ ID NOs:394-401, 406-409, 438-441,or 446-449.

In any of the aspects and embodiments provided herein that include, oroptionally include, a nucleic acid sequence encoding an inhibitory RNAmolecule, including, but not limited to, aspects and embodimentsprovided in this Exemplary Embodiments section, unless already statedtherein, or incompatible therewith, such nucleic acid sequence isincluded and such inhibitory RNA molecule, in certain embodiments,include 2 or more, 2-10, 2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8inhibitory RNA, or of the targeted inhibitory RNA (e.g. miRNA)identified herein, for example in the paragraph immediately above; or incertain embodiments such polynucleotide includes 2 or more, 2-10, 2-8,2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g. miRNA) that targetmRNA encoding FAS, cCBL, AHR, CD3z, Cbx, HOMES, or HK2, or a combinationof 1 or more inhibitory RNA that target such mRNA; or in certain furtherillustrative embodiments, such polynucleotide includes 2 or more, 2-10,2-8, 2-6, 3-5, 2, 3, 4, 5, 6, 7, or 8 inhibitory RNA (e.g. miRNA) thattarget mRNA encoding FAS, AHR, Cbx3, HOMES, or HK2, or a combination of1 or more inhibitory RNA that target such mRNA. Such aspects andembodiments provided herein that include a nucleic acid that encodes aninhibitory RNA molecule, include, but are not limited to, aspects andembodiments provided herein that are directed to polynucleotides orvectors, for example replication incompetent retroviral particles, oraspects comprising a genome, such as isolated cells or replicationincompetent retroviral particles.

Provided herein in one aspect is a method for genetically modifyingand/or transducing a lymphocyte (e.g. a T cell or an NK cell) or apopulation thereof, comprising contacting blood cells comprising thelymphocyte (e.g. the T cell or NK cell) or the population thereof, exvivo with a replication incompetent recombinant retroviral particlecomprising in its genome a polynucleotide comprising one or more nucleicacid sequences operatively linked to a promoter active in lymphocytes(e.g. T cells and/or NK cells), wherein a first nucleic acid sequence ofthe one or more nucleic acid sequences encodes a chimeric antigenreceptor (CAR) comprising an antigen-specific targeting region (ASTR), atransmembrane domain, and an intracellular activating domain, andoptionally another of the one or more nucleic acid sequences encodes oneor more (e.g. two or more) inhibitory RNA molecules directed against oneor more RNA targets, and further optionally another of the one or morenucleic acid sequences encodes a polypeptide lymphoproliferativeelement, wherein said contacting facilitates genetic modification and/ortransduction of the lymphocyte (e.g. T cell or NK cell), or at leastsome of the lymphocytes (e.g. T cells and/or NK cells) by thereplication incompetent recombinant retroviral particle, therebyproducing a genetically modified and/or transduced lymphocyte (e.g. Tcell and/or NK cell). In such method, the contacting is typicallyperformed in a reaction mixture, sometimes referred to herein as atransduction reaction mixture, comprising a population of lymphocytes(e.g. T cells and/or NK cells) and contacted with a population ofreplication incompetent recombinant retroviral particles. Variouscontacting times are provided herein, including, but not limited to, inthis Exemplary Embodiments section, that can be used in this aspect tofacilitate membrane association, and eventual membrane fusion of thelymphocytes (e.g. T cells and/or the NK cells) to the replicationincompetent recombinant retroviral particles. In an illustrativeembodiment contacting is performed for less than 15 minutes.

Provided herein in one aspect, is use of replication incompetentrecombinant retroviral particles in the manufacture of a kit forgenetically modifying lymphocytes (e.g. T cells or NK cells) of asubject, wherein the use of the kit comprises: contacting blood cellscomprising the lymphocytes (e.g. T cells and/or the NK cells) ex vivo ina reaction mixture, with the replication incompetent recombinantretroviral particles, wherein the replication incompetent recombinantretroviral particles comprise a pseudotyping element on their surface,wherein the replication incompetent recombinant retroviral particlescomprise a polynucleotide comprising one or more nucleic acid sequences,typically transcriptional units operatively linked to a promoter activein lymphocytes (e.g. T cells and/or NK cells), wherein the one or moretranscriptional units encode a first polypeptide comprising a chimericantigen receptor (CAR), a first polypeptide comprising alymphoproliferative element (LE), or a first polypeptide comprising anLE and a second polypeptide comprising a CAR, thereby producing thegenetically modified lymphocytes (e.g. the genetically modified T cellsand/or the genetically modified NK cells). Various contacting times areprovided herein, including, but not limited to, in this ExemplaryEmbodiments section, that can be used in this aspect to facilitatemembrane association, and eventual membrane fusion of the lymphocytes(e.g. T cells and/or the NK cells) to the replication incompetentrecombinant retroviral particles. In an illustrative embodiment,contacting is performed for less than 15 minutes.

In another aspect, provided herein is a genetically modified lymphocyte(e.g. T cell or NK cell) made by genetically modifying lymphocytes (e.g.T cells and/or NK cells) according to a method comprising contactingblood cells comprising the T cells or NK cells ex vivo in a reactionmixture, with replication incompetent recombinant retroviral particles,wherein the replication incompetent recombinant retroviral particlescomprise a pseudotyping element on their surface, wherein thereplication incompetent recombinant retroviral particles comprise apolynucleotide comprising one or more nucleic acid sequences, typicallytranscriptional units operatively linked to a promoter active inlymphocytes (e.g. T cells and/or NK cells), wherein the one or moretranscriptional units encode a first polypeptide comprising a chimericantigen receptor (CAR), a first polypeptide comprising alymphoproliferative element (LE), or a first polypeptide comprising anLE and a second polypeptide comprising a CAR, thereby producing thegenetically modified lymphocytes (e.g. T cells and/or the geneticallymodified NK cells). Various contacting times are provided herein,including, but not limited to, in this Exemplary Embodiments section,that can be used in this aspect to facilitate membrane association, andeventual membrane fusion of the lymphocytes (e.g. T cells and/or the NKcells) to the replication incompetent recombinant retroviral particles.In an illustrative embodiment, contacting is performed for less than 15minutes.

Provided herein in another aspect is a replication incompetentrecombinant retroviral particle for use in a method for geneticallymodifying lymphocyte, for example a T cell and/or NK cell, wherein themethod comprises contacting blood cells comprising the lymphocyte, forexample T cell and/or NK cell, of the subject in a reaction mixture, exvivo, with a replication incompetent recombinant retroviral particlecomprising in its genome a polynucleotide comprising one or more nucleicacid sequences operatively linked to a promoter active in T cells and/orNK cells, wherein a first nucleic acid sequence of the one or morenucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, and optionally anotherof the one or more nucleic acid sequences encodes one or more (e.g. twoor more) inhibitory RNA molecules directed against one or more RNAtargets, and further optionally another of the one or more nucleic acidsequences encodes a polypeptide lymphoproliferative element, whereinsaid contacting facilitates transduction of at least some of the restingT cells and/or NK cells by the replication incompetent recombinantretroviral particles, thereby producing a genetically modified T celland/or NK cell. Various contacting times are provided herein, including,but not limited to, in this Exemplary Embodiments section, that can beused in this aspect to facilitate membrane association, and eventualmembrane fusion of the lymphocytes (e.g. T cells and/or the NK cells) tothe replication incompetent recombinant retroviral particles. In anillustrative embodiment, contacting is performed for less than 15minutes. In some embodiments the method can further include introducingthe genetically modified T cell and/or NK cell into a subject. Inillustrative embodiments, the blood cells comprising the lymphocyte(e.g. the T cell and/or NK cell) are from the subject, and thus theintroducing is a reintroducing. In this aspect, in some embodiments, apopulation of lymphocytes (e.g. T cells and/or NK cells) are contactedin the contacting step, genetically modified and/or transduced, andintroduced into the subject in the introducing step.

Provided herein in another aspect is the use of a replicationincompetent recombinant retroviral particle in the manufacture of a kitfor genetically modifying a lymphocyte, for example a T cell and/or NKcell of a subject, wherein the use of the kit comprises contacting bloodcells comprising the lymphocyte, for example the T cell and/or the NKcell of the subject ex vivo in a reaction mixture, with replicationincompetent recombinant retroviral particles comprising in their genomea polynucleotide comprising one or more nucleic acid sequencesoperatively linked to a promoter active in T cells and/or NK cells,wherein a first nucleic acid sequence of the one or more nucleic acidsequences encodes a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region (ASTR), a transmembrane domain, and anintracellular activating domain, and optionally another of the one ormore nucleic acid sequences encodes one or more (e.g. two or more)inhibitory RNA molecules directed against one or more RNA targets, andfurther optionally another of the one or more nucleic acid sequencesencodes a polypeptide lymphoproliferative element, wherein saidcontacting facilitates genetic modification of at least some of the Tcells and/or NK cells by the replication incompetent recombinantretroviral particles, thereby producing a genetically modified T celland/or NK cell. As indicated herein, various contacting times areprovided herein, that can be used in this aspect to facilitate membraneassociation, and eventual membrane fusion of the lymphocyte (e.g. T celland/or the NK cell) to the replication incompetent recombinantretroviral particles. In an illustrative embodiment, contacting isperformed for less than 15 minutes. In illustrative embodiments, theblood cells comprising the lymphocyte (e.g. the T cell and/or NK cell)are from the subject, and thus the introducing is a reintroducing. Inthis aspect, in some embodiments, a population of T cells and/or NKcells are contacted in the contacting step, genetically modified and/ortransduced, and introduced into the subject in the introducing step.

Provided herein in another aspect is the use of replication incompetentrecombinant retroviral particles in the manufacture of a medicament forgenetically modifying lymphocytes, for example T cells and/or NK cellsof a subject, wherein the use of the medicament comprises:

-   -   A) contacting blood cells comprising the T cells and/or NK cells        of the subject ex vivo in a reaction mixture, with the        replication incompetent recombinant retroviral particles        comprising in their genome a polynucleotide comprising one or        more nucleic acid sequences operatively linked to a promoter        active in T cells and/or NK cells, wherein a first nucleic acid        sequence of the one or more nucleic acid sequences encodes a        chimeric antigen receptor (CAR) comprising an antigen-specific        targeting region (ASTR), a transmembrane domain, and an        intracellular activating domain, and optionally another of the        one or more nucleic acid sequences encodes one or more (e.g. two        or more) inhibitory RNA molecules directed against one or more        RNA targets, and further optionally another of the one or more        nucleic acid sequences encodes a polypeptide lymphoproliferative        element, wherein said contacting facilitates genetic        modification of at least some of the lymphocytes (for example, T        cells and/or NK cells) by the replication incompetent        recombinant retroviral particles, thereby producing genetically        modified T cells and/or NK cells; and optionally    -   B) introducing the genetically modified T cell and/or NK cell        into the subject, thereby genetically modifying the lymphocytes,        for example T cells and/or NK cells of the subject.

In such aspects in the immediately above paragraph, as indicated herein,various contacting times are provided herein, that can be used in thisaspect to facilitate membrane association, and eventual membrane fusionof the lymphocytes (e.g. T cells and/or the NK cells) to the replicationincompetent recombinant retroviral particles. In an illustrativeembodiment, contacting is performed for less than 15 minutes. In someembodiments of such method, the blood cells, lymphocyte(s) (e.g. Tcell(s) and/or NK cell(s)) are from a subject, typically in suchembodiments from blood collected from the subject. In some embodimentsof the method aspect provided in this paragraph, the geneticallymodified and/or transduced lymphocyte (e.g. T cell and/or NK cell) orpopulation thereof, is introduced or reintroduced into a subject.

In any of the use aspects herein, genetically modified lymphocyte(s)(e.g. T cell(s) or NK(s) cell) aspects herein, or methods aspects forgenetically modifying and/or transducing a lymphocyte(s) (e.g. T cell(s)or an NK cell(s)) according to any embodiment herein, including but notlimited to, any embodiment in this Exemplary Embodiments section,including those above, unless incompatible with, or already stated, thereaction mixture comprises at least 10%, 20%, 25%, 50%, 75%, 80%, 90%,95%, or 99% whole blood and optionally an effective amount of ananticoagulant, or the reaction mixture further comprises at least oneadditional blood or blood preparation component that is not a PBMC, andin further illustrative embodiments such blood or blood preparationcomponent is one or more of the Noteworthy Non-PBMC Blood or BloodPreparation Components provided herein.

In another aspect, provided herein is a reaction mixture, comprisingreplication incompetent recombinant retroviral particles, a T cellactivation element, and blood cells, wherein the recombinant retroviralparticles comprise a pseudotyping element on their surface, wherein theblood cells comprise T cells and/or NK cells, wherein the replicationincompetent recombinant retroviral particles comprise a polynucleotidecomprising one or more nucleic acid sequences, typically transcriptionalunits operatively linked to a promoter active in T cells and/or NKcells, wherein the one or more transcriptional units encode a firstpolypeptide comprising a chimeric antigen receptor (CAR), a firstpolypeptide comprising a lymphoproliferative element (LE), and/or one ormore inhibitory RN A molecules, and wherein the reaction mixturecomprises at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, or 99% wholeblood. The one or more inhibitory RNA molecule(s) can be directedagainst any target provided herein, including, but not limited to, inthis Exemplary Embodiments section.

In one aspect, provided herein is a reaction mixture, comprisingreplication incompetent recombinant retroviral particles, and bloodcells, wherein the recombinant retroviral particles comprise apseudotyping element on their surface, wherein the blood cells compriseT cells and/or NK cells, and wherein the reaction mixture comprises atleast 10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% wholeblood and optionally an effective amount of an anticoagulant, or whereinthe reaction mixture further comprises at least one additional blood orblood preparation component that is not a PBMC, and in illustrativeembodiments such blood or blood preparation component is one or more ofthe Noteworthy Non-PBMC Blood or Blood Preparation Components providedherein.

In another aspect, provided herein is a reaction mixture, comprisingreplication incompetent recombinant retroviral particles, a T cellactivation element, and blood cells, wherein the recombinant retroviralparticles comprise a pseudotyping element on their surface, wherein theblood cells comprise T cells and/or NK cells, wherein the replicationincompetent recombinant retroviral particles comprise a polynucleotidecomprising one or more nucleic acid sequences, typically transcriptionalunits operatively linked to a promoter active in T cells and/or NKcells, wherein the one or more transcriptional units encode a firstpolypeptide comprising a chimeric antigen receptor (CAR), a firstpolypeptide comprising a lymphoproliferative element (LE), and/or one ormore inhibitory RNA molecules, and wherein the reaction mixturecomprises at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, 95%, or 99% wholeblood and optionally an effective amount of an anticoagulant, or whereinthe reaction mixture further comprises at least one additional blood orblood preparation component that is not a PBMC, and in illustrativeembodiments such blood or blood preparation component is one or more ofthe Noteworthy Non-PBMC Blood or Blood Preparation Components providedherein. The one or more inhibitory RNA molecule(s) can be directedagainst any target provided herein, including, but not limited to, inthis Exemplary Embodiments section.

In another aspect, provided herein is a method for genetically modifyingT cells and/or NK cells in blood or a component thereof, comprisingcontacting blood cells comprising tire T cells and/or NK cells ex vivo,with replication incompetent recombinant retroviral particles in areaction mixture, wherein the replication incompetent recombinantretroviral particles comprise a pseudotyping element on their surface,wherein said contacting facilitates association of the T cells and/or NKcells with the replication incompetent recombinant retroviral particles,wherein the recombinant retroviral particles genetically modify and/ortransduce the T cells and/or NK cells, and wherein the reaction mixturecomprises at least 10%, 10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%,95%, or 99% whole blood and optionally an effective amount of ananticoagulant, or wherein the reaction mixture further comprises atleast one additional blood or blood preparation component that is not aPBMC, and in illustrative embodiments such blood or blood preparationcomponent is one or more of the Noteworthy Non-PBMC Blood or BloodPreparation Components provided herein

In another aspect, provided herein is use of replication incompetentrecombinant retroviral particles in the manufacture of a kit forgenetically modifying T cells and/or NK cells of a subject wherein theuse of the kit comprises: contacting blood cells comprising the T cellsand/or NKs cell ex vivo in a reaction mixture, with the replicationincompetent recombinant retroviral particles, wherein the replicationincompetent recombinant retroviral particles comprise a pseudotypingelement on their surface, wherein said contacting facilitatesassociation of the T cells or NK cells with the replication incompetentrecombinant retroviral particles, wherein the recombinant retroviralparticles genetically modify and/or transduce the T cells and/or NKcells, and wherein the blood cells comprise T cells, NK cells, andwherein the reaction mixture comprises at least 10%, 20%, 25%, 50%, 60%,70%, 75%, 80%, 90%, 95%, or 99% whole blood and optionally an effectiveamount of an anticoagulant, or wherein the reaction mixture furthercomprises at least one additional blood or blood preparation componentthat is not a PBMC, and in illustrative embodiments such blood or bloodpreparation component is one or more of the Noteworthy Non-PBMC Blood orBlood Preparation Components provided herein.

In another aspect, provided herein is a genetically modified T cell orNK cell made by genetically modifying T cells and/or NK cells accordingto a method comprising, contacting blood cells comprising the T cellsand/or NK cells ex vivo, with replication incompetent recombinantretroviral particles in a reaction mixture, wherein the replicationincompetent recombinant retroviral particles comprise a pseudotypingelement on their surface, wherein said contacting facilitatesassociation of the T cells and/or NK cells with the replicationincompetent recombinant retroviral particles, wherein the recombinantretroviral particles genetically modify and/or transduce the T cellsand/or NK cells, and wherein the reaction mixture comprises at least10%, 20%, 25%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% whole blood andoptionally an effective amount of an anticoagulant, or wherein thereaction mixture further comprises at least one additional blood orblood preparation component that is not a PBMC, and in illustrativeembodiments such blood or blood preparation component is one or more ofthe Noteworthy Non-PBMC Blood or Blood Preparation Components providedherein.

The one or more Noteworthy Non-PBMC Blood or Blood PreparationComponents are present in certain illustrative embodiments of any of thereaction mixture, use, genetically modified T cell or NK cell, or methodfor genetically modifying T cells and/or NK cells provided herein,including but not limited to those provided in this ExemplaryEmbodiments section, because in these certain illustrative embodiments,the reaction mixture comprises at least 10% whole blood. In certainembodiments of any of the reaction mixture, use, genetically modified Tcell or NK cell, or method for genetically modifying T cells and/or NKcells provided herein, included but not limited to those provided inthis Exemplary Embodiments section, unless incompatible with, or alreadystated in an aspect or embodiment the reaction mixture comprises between10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% and 75% on the low end ofthe range, and 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% on thehigh end of the range of whole blood, or at least 10%, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 96% 97%, 98%, 99%, 99.9%,or 99.99% whole blood.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the blood cells inthe reaction mixture comprise at least 10% neutrophils and at least 0.5%eosinophils, as a percent of the white blood cells in the reactionmixture.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture comprises at least 20%, 25%, 30% or 40% neutrophils as a percentof white blood cells in the reaction mixture, or between 20% and 80%,25% and 75%, or 40% and 60% neutrophils as a percent of white bloodcells in the reaction mixture.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture comprises at least 0.1% eosinophils, or between 0.25% and 8%eosinophils, or between 0.5% and 4% as a percent of white blood cells inthe reaction mixture.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the blood cells inthe reaction mixture are not subjected to a PBMC enrichment procedurebefore the contacting.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture is formed by adding the recombinant retroviral particles towhole blood.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture is formed by adding the recombinant retroviral particles tosubstantially whole blood comprising an effective amount of ananti-coagulant.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture is in a closed cell processing system. In certain embodiments ofsuch a reaction mixture, use, genetically modified T cell or NK cell, ormethod for genetically modifying T cells and/or NK cells, the bloodcells in a reaction mixture are PBMCs and the reaction mixture is incontact with a leukodepletion filter assembly in the closed cellprocessing system, and in optional further embodiments theleukodepletion filter assembly comprises a HemaTrate filter.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture comprises an anti-coagulant. For example, in certainembodiments, the anti-coagulant is selected from the group consisting ofacid citrate dextrose, EDTA, or heparin. In certain embodiments, theanti-coagulant is other than acid citrate dextrose. In certainembodiments, the anti-coagulant comprises an effective amount ofheparin.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture is in a blood bag during the contacting.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture is in contact with a T lymphocyte and/or NK cell-enrichingfilter in the closed cell processing system before the contacting, andwherein the reaction mixture comprises granulocytes, wherein thegranulocytes comprise at least 10% of the white blood cells in thereaction mixture, or wherein the reaction mixture comprises at least 10%as many granulocytes as T cells, wherein the genetically modifiedlymphocytes (e.g. T cells or NK cells) are subject to a PBMC enrichmentprocess after the contacting.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, blood cells in thereaction mixture are PBMCs and wherein the reaction mixture is incontact with a leukodepletion filter assembly in the closed cellprocessing system after the contacting comprising an optional incubatingin the reaction mixture.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the whole blood isother than cord blood.

In certain embodiments of any of the reaction mixture, use, geneticallymodified T cell or NK cell, or method for genetically modifying T cellsand/or NK cells provided herein, included but not limited to thoseprovided in this Exemplary Embodiments section, unless incompatiblewith, or already stated in an aspect or embodiments, the reactionmixture is in contact with a leukodepletion filter assembly in a closedcell processing system before the contacting, at the time therecombinant retroviral particles and the blood cells are contacted,during the contacting comprising an optional incubating in the reactionmixture, and/or after the contacting comprising the optional incubatingin the reaction mixture, wherein the T cells and/or NK cells, or thegenetically modified T cells and/or NK cells are further subjected to aPBMC enrichment procedure.

In one aspect, provided herein is a replication incompetent recombinantretroviral particle comprising in its genome a polynucleotide comprisingone or more nucleic acid sequences operatively linked to a promoteractive in T cells and/or NK cells, wherein:

-   -   a. a first nucleic acid sequence of the one or more nucleic acid        sequences encodes one or more (e.g. two or more) inhibitory RNA        molecules directed against one or more RNA targets, and    -   b. a second nucleic acid sequence of the one or more nucleic        acid sequences encodes a chimeric antigen receptor (CAR)        comprising an antigen-specific targeting region (ASTR), a        transmembrane domain, and an intracellular activating domain.        The one or more inhibitory RNA molecule(s) can be directed        against any target provided herein, including, but not limited        to, in this Exemplary Embodiments section.

Provided in another aspect herein is a mammalian packaging cell linecomprising a packageable RNA genome for a replication incompetentretroviral particle, wherein said packageable RNA genome comprises:

-   -   a. a 5′ long terminal repeat, or active fragment thereof;    -   b. a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   c. a polynucleotide comprising one or more nucleic acid        sequences operatively linked to a promoter active in T cells        and/or NK cells, wherein a first nucleic acid sequence of the        one or more nucleic acids encodes one or more (e.g. two or more)        inhibitory RNA molecules directed against one or more RNA        targets and a second nucleic acid sequence of the one or more        nucleic acid sequences encodes a chimeric antigen receptor (CAR)        comprising an antigen-specific targeting region (ASTR), a        transmembrane domain, and an intracellular activating domain;        and    -   d. a 3′ long terminal repeat, or active fragment thereof. The        one or more inhibitory RNA molecule(s) can be directed against        any target provided herein, including, but not limited to, in        this Exemplary Embodiments section.

Provided in another aspect herein is a retroviral vector comprising apackageable RNA genome for a replication incompetent retroviralparticle, wherein said packageable RNA genome comprises:

-   -   a) a 5′ long terminal repeat or active fragment thereof;    -   b) a nucleic acid sequence encoding a retroviral cis-acting RNA        packaging element;    -   c) a polynucleotide comprising one or more nucleic acid        sequences operatively linked to a promoter active in T cells        and/or NK cells, wherein a first nucleic acid sequence of the        one or more nucleic acids encodes one or more (e.g. two or more)        inhibitory RNA molecules directed against one or mote RNA        targets and a second nucleic acid sequence of the one or mote        nucleic acid sequences encodes a chimeric antigen receptor (CAR)        comprising an antigen-specific targeting region (ASTR), a        transmembrane domain, and an intracellular activating domain;        and    -   d) a 3′ long terminal repeat or active fragment thereof. The one        or mote inhibitory RNA molecule(s) can be directed against any        target provided herein, including, but not limited to, in this        Exemplary Embodiments section.

In some embodiments of the retroviral vector aspect or the mammalianpackaging cell line aspect the polynucleotide of (c) can be in reverseorientation to the nucleic acid sequence encoding tire retroviralcis-acting RNA packaging element (b), the 5′ long terminal repeat (a),and/or the 3′ long terminal repeat (d).

In some embodiments of the retroviral vector aspect or the mammalianpackaging cell line aspect, expression of the packageable RNA genome isdriven by an inducible promoter active in the mammalian packaging cellline.

In some embodiments of the retroviral vector aspect or the mammalianpackaging cell line aspect, the retroviral cis-acting RNA packagingelement can comprise a central polypurine tract (cPPT)/centraltermination sequence, an HIV Psi, or a combination thereof. Theretroviral vector can optionally include an antibiotic resistance geneand/or a detectable marker.

Provided herein in another aspect is a genetically modified T celland/or NK cell comprising:

a) one or more (e.g. two or more) inhibitory RNA molecules directedagainst one or more RNA targets; and

b) a chimeric antigen receptor (CAR) comprising an antigen-specifictargeting region (ASTR), a transmembrane domain, and an intracellularactivating domain, wherein said one or more (e.g. two or more)inhibitory RNA molecules and the CAR are encoded by nucleic acidsequences that are genetic modifications of the T cell and/or NK cell.The one or more inhibitory RNA molecule(s) can be directed against anytarget provided herein, including, but not limited to, in this ExemplaryEmbodiments section.

In some embodiments of the genetically modified T cell and/or NK cellaspect, the genetically modified T cell and/or NK cell also comprises atleast one lymphoproliferative element that is not an inhibitory RNAmolecule, typically a polypeptide lymphoproliferative element, whereinsaid lymphoproliferative element is encoded by a nucleic acid that is agenetic modification of the T cell and/or NK cell. In some embodiments,the inhibitory RNA molecules, the CAR, and/or the at least onepolypeptide lymphoproliferative element are expressed in a polycistronicmatter. In illustrative embodiments, the inhibitory RNA molecules areexpressed from a single polycistronic transcript.

Provided herein in another aspect is a replication incompetentrecombinant retroviral particle, wherein the replication incompetentrecombinant retroviral particle comprises in its genome a polynucleotidecomprising one or more nucleic acid sequences operatively linked to apromoter active in T cells and/or NK cells, wherein a first nucleic acidsequence of the one or more nucleic acid sequences encodes one or more(e.g. two or more) inhibitory RNA molecules directed against one or moreRNA targets and a second nucleic acid sequence of the one or morenucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, wherein the methodcomprises contacting a T cell and/or NK cell of the subject ex vivo, andsaid contacting facilitates transduction of at least some of the restingT cells and/or NK cells by the replication incompetent recombinantretroviral particles, thereby producing a genetically modified T celland/or NK cell. The one or more inhibitory RNA molecule(s) can bedirected against any target provided herein, including, but not limitedto, in this Exemplary Embodiments section.

Provided herein in another aspect is a commercial container containing areplication incompetent recombinant retroviral particle and optionallyinstructions for the use thereof to treat tumor growth in a subjectwherein the replication incompetent recombinant retroviral particlecomprises in its genome a polynucleotide comprising one or more nucleicacid sequences operatively linked to a promoter active in T cells and/orNK cells, wherein a first nucleic acid sequence of the one or morenucleic acid sequences encodes one or more (e.g. two or more) inhibitoryRNA molecules directed against one or more RNA targets and a secondnucleic acid sequence of the one or more nucleic acid sequences encodesa chimeric antigen receptor (CAR) comprising an antigen-specifictargeting region (ASTR), a transmembrane domain, and an intracellularactivating domain. The one or more inhibitory RNA molecule(s) can bedirected against any target provided herein, including, but not limitedto, in this Exemplary Embodiments section.

In any of the aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, and a second nucleic acid sequence of the oneor more nucleic acid sequences encodes a chimeric antigen receptor (CAR)comprising an antigen-specific targeting region (ASTR), a transmembranedomain, and an intracellular activating domain, the polynucleotide mayfurther include a third nucleic acid sequence that encodes at least onelymphoproliferative element that is not an inhibitory RNA molecule, andin illustrative embodiments is a polypeptide, for example any of thepolypeptide lymphoproliferative elements disclosed herein.

In any of the aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, the inhibitory RNA molecule can have any of thestructures and/or be any of the embodiments provided herein in theInhibitory RNA Molecules section. For example, the inhibitory RNA can insome embodiments include a 5′ strand and a 3′ strand that are partiallyor fully complementary to one another, wherein said 5′ strand and said3′ strand are capable of forming an 18-25 nucleotide RNA duplex.Furthermore, the inhibitory RNA molecule can be a miRNA or an shRNA andin certain embodiments, at least one or all of the inhibitory RNAmolecules comprise a 5′ arm, 3′ arm, or both, derived from a naturallyoccurring miRNA. For example, such as a naturally occurring miRNA can beselected from the group consisting of: miR-155, miR-30, miR-17-92,miR-122, and miR-21, and in illustrative embodiments miR-155.

In any of the aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of the one or more nucleic acid sequences encodestwo or more inhibitory RNA molecules directed against one or more RNAtargets, in some embodiments, the first nucleic acid sequence encodestwo to four inhibitory RNA molecules. In illustrative embodiments,between 2 and 10, 2 and 8, 2 and 6, 2 and 5, 2 and 4, 3 and 5, or 3 and6 inhibitory RNA molecules are included in the first nucleic acidsequence. In an illustrative embodiment, four inhibitory RNA moleculesare included in the first nucleic acid sequence.

In any of the aspects provided immediately above that include apolynucleotide comprising one or more nucleic acid sequences operativelylinked to a promoter active in T cells and/or NK cells, wherein a firstnucleic acid sequence of tire one or more nucleic acid sequences encodesone or more (e.g. two or more) inhibitory RNA molecules directed againstone or more RNA targets, the one or more (e.g. two or more) inhibitoryRNA molecules can be in an intron. In some embodiments, the intron is ina promoter. In illustrative embodiments, the intron is EF-1alpha intronA. In some embodiments, the intron is adjacent to and downstream of apromoter, which in illustrative embodiments, is inactive in a packagingcell used to produce the replication incompetent recombinant retroviralparticle.

In any of the reaction mixture, use, genetically modified T cell or N Kcell, or method for genetically modifying T cells and/or NK cellsaspects and embodiments provided herein, including, but not limited to,in this Exemplary Embodiments section, unless incompatible with, orotherwise stated at least 10%, 20%, 25%, 30%, 40%, 50%, most, 60%, 70%,75%, 80%, 90%, 95%, or 99% of the T cells are resting T cells, or of theNK cells are resting NK cells, when they are combined with thereplication incompetent retroviral particles to form the reactionmixture.

In any of the use, genetically modified T cell or NK cell, or method forgenetically modifying T cells and/or NK cells aspects and embodimentsprovided herein, including, but not limited to, in this ExemplaryEmbodiments section, unless incompatible with, or otherwise stated, thecell or cells are not subjected to a spinoculation procedure, forexample not subjected to a spinoculation of at least 800 g for at least30 minutes.

In some embodiments of any of the use, genetically modified T cell or NKcell, or method for genetically modifying T cells and/or NK cellsaspects and embodiments provided herein, including, but not limited to,in this Exemplary Embodiments section, unless incompatible with, orotherwise stated, the method further comprises administering thegenetically modified T cells and/or NK cells to a subject, optionallywherein the subject is the source of the blood cells. In somesubembodiments of these and embodiments of any of the methods and usesherein, including those in this Exemplary Embodiments section, providedthat it is not incompatible with, or already stated, the geneticallymodified and/or transduced lymphocyte (e.g. T cell and/or NK cell) orpopulation thereof, undergoes 4 or fewer cell divisions ex vivo prior tobeing introduced or reintroduced into the subject. In some embodiments,no more than 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour pass(es)between the time blood is collected from the subject and the time thegenetically modified T cells and/or NK cells are reintroduced into thesubject. In some embodiments, all steps after the blood is collected andbefore the blood is reintroduced, are performed in a closed system,optionally in which a person monitors the closed system throughout theprocessing.

In any of the replication incompetent recombinant retroviral particle,reaction mixture, use, genetically modified T cell or NK cell, or methodfor genetically modifying T cells and/or NK cells aspects andembodiments provided herein, including, but not limited to, in thisExemplary Embodiments section, unless incompatible with, or otherwisestated, the replication incompetent recombinant retroviral particle(s)comprise a membrane-bound T cell activation element on their surface. Insome subembodiments of these and embodiments of any of the aspectsprovided herein, including those in this Exemplary Embodiments section,provided that it is not incompatible with, or already stated, the T cellactivation element can be one or more of an anti-CD3 antibody or ananti-CD28 antibody. In some embodiments of these and embodiments of anyof the aspects provided herein, including, but not limited to, in thisExemplary Embodiments section, unless incompatible with, or otherwisestated, the T cell activation element is one or more polypeptides, inillustrative embodiments membrane-bound polypeptides capable of bindingCD28, OX40, 4-1BB, ICOS, CD9, CD53, CD63, CD81, and/or CD82. In someembodiments, a membrane-bound polypeptide capable of binding to CD3 isfused to a heterologous GPI anchor attachment sequence and/or amembrane-bound polypeptide capable of binding to CD28 is fused to aheterologous GPI anchor attachment sequence. In illustrativeembodiments, the membrane-bound polypeptide capable of binding to CD28is CD80, or an extra-cellular domain thereof, bound to a CD16B GPIanchor attachment sequence. In some embodiments, the T cell activationelement further includes one or more polypeptides capable of bindingCD3. In some subembodiments of these and embodiments of any of theaspects provided herein, including those in this Exemplary Embodimentssection, provided that it is not incompatible with, or already-stated,the T cell activation element is a membrane-bound anti-CD3 antibody,wherein the anti-CD3 antibody is bound to the membrane of therecombinant retroviral particles. In some embodiments, themembrane-bound anti-CD3 antibody is anti-CD3 scFv or an anti-CD3 scFvFc.In some embodiments, the membrane-bound anti-CD3 antibody is bound tothe membrane by a heterologous GPI anchor. In some embodiments, theanti-CD3 antibody is a recombinant fusion protein with a viral envelopeprotein. In some embodiments, the anti-CD3 antibody is a recombinantfusion protein with the viral envelope protein from MuLV. In someembodiments, the anti-CD3 is a recombinant fusion protein with the viralenvelope protein of MulV which is mutated at a furin cleavage site.

In any of the use, genetically modified T cell or NK cell, or method forgenetically modifying T cells and/or NK cells aspects and embodimentsprovided herein, including, but not limited to, in this ExemplaryEmbodiments section, unless incompatible with, or otherwise stated, anABC transporter inhibitor and/or substrate, in further subembodiments anexogenous ABC transporter inhibitor and/or substrate, is not presentbefore, during, or both before and during the genetic modificationand/or transduction.

In any of the reaction mixture, use, genetically modified T cell or NKcell, or method for genetically modifying T cells and/or NK cellsaspects and embodiments provided herein, including, but not limited to,in this Exemplary Embodiments section, unless incompatible with, orotherwise stated, the recombinant retroviral particles are present inthe reaction mixture at an MOI of between 0.1 and 50, 0.5 and 50, 0.5and 20, 0.5 and 10, 1 and 25, 1 and 15, 1 and 10, 1 and 5, 2 and 15, 2and 10, 2 and 7, 2 and 3, 3 and 10, 3 and 15, or 5 and 15 or at least0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15 or are present in the reactionmixture at an MOI of at least 0.1, 0.5, 1, 2, 2.5, 3, 5, 10 or 15.

In any of the reaction mixture, use, genetically modified T cell or NKcell, or method for genetically modifying T cells and/or NK cellsaspects and embodiments provided herein, including, but not limited to,in this Exemplary Embodiments section, unless incompatible with, orotherwise stated, at least 5%, at least 10%, at least 15%, or at least20% of the T cells and/or NK cells are genetically modified, or between5%, 10%, 15%, 20%, or 25% on the low end of the range, and 20%, 25%,50%, 60%, 70%, 80%, or 85% on the high end of the range.

In any of the polynucleotide, replication incompetent recombinantretroviral particle, reaction mixture, use, genetically modified T cellor NK cell, or method for genetically modifying T cells and/or NK cellsaspects and embodiments provided herein, including, but not limited to,in this Exemplary Embodiments section, unless incompatible with, orotherwise stated, the one or more transcriptional units can encode apolypeptide comprising a lymphoproliferative element (LE). Any of thepolypeptide lymphoproliferative elements disclosed herein, for example,but not limited to those disclosed in the “Lymphoproliferative elements”section herein, or functional mutants and/or fragments thereof, can beencoded. In some embodiments, the LE comprises an intracellular domainfrom CD2, CD3D, CD3E, CD3G, CD4, CD8A, CD8B, CD27, mutated Delta LckCD28, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR,FCER1G, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2,IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R,IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD,IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA,IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8,TNFRSF9, TNFRSF14, or TNFRSF18, or functional mutants and/or fragmentsthereof.

In any of the replication incompetent recombinant retroviral particle,reaction mixture, use, genetically modified T cell or NK cell, or methodfor genetically modifying T cells and/or NK cells aspects andembodiments provided herein, including, but not limited to, in thisExemplary Embodiments section, unless incompatible with, or otherwisestated, the replication incompetent recombinant retroviral particles arelentiviral particles. In further illustrative embodiments, thegenetically modified cell is a genetically modified T cell or agenetically modified NKT cell.

In any of the polynucleotide, replication incompetent recombinantretroviral particle, reaction mixture, use, genetically modified T cellor NK cell, or method for genetically modifying T cells and/or NK cellsaspects and embodiments provided herein, including, but not limited to,in this Exemplary Embodiments section, unless incompatible with, orotherwise stated, the one or more transcriptional units can encode apolypeptide comprising a CAR. In some embodiments, the CAR is amicroenvironment restricted biologic (MRB)-CAR. In other embodiments,the ASTR of the CAR binds to a tumor associated antigen. In otherembodiments, the ASTR of the CAR is a microenvironment-restrictedbiologic (MRB)-ASTR.

In certain embodiments, any of the aspects and embodiments providedherein that include a polynucleotide, in some instances in the genome ofa replication incompetent recombinant retroviral particle or agenetically modified T cell and/or NK cell, that comprises a nucleicacid sequences operatively linked to a promoter active in T cells and/orNK cells, that encodes at least one polypeptide lymphoproliferativeelement. In illustrative embodiments, the polypeptidelymphoproliferative element is any of the polypeptidelymphoproliferative elements disclosed herein. In some embodiments, anyor all of the nucleic acid sequences provided herein can be operablylinked to a riboswitch. In some embodiments, the riboswitch is capableof binding a nucleoside analog. In some embodiments, the nucleosideanalog is an antiviral drug.

In any of the aspects and embodiments provided herein that include areplication incompetent recombinant retroviral particle, including, butnot limited to aspects and embodiments in this Exemplary Embodimentssection, unless incompatible with, or already stated in an aspect orembodiment, in illustrative embodiments, the replication incompetentrecombinant retroviral particle comprises a pseudotyping element on itssurface that is capable of binding to a T cell and/or NK cell andfacilitating membrane fusion of the replication incompetent recombinantretroviral particle thereto. In some embodiments, the pseudotypingelement is a viral envelope protein. In some embodiments, the viralenvelope protein is one or more of the feline endogenous virus (RD114)envelope protein, the oncoretroviral amphotropic envelope protein, theoncoretroviral ecotropic envelope protein, the vesicular stomatitisvirus envelope protein (VSV-G), the baboon retroviral envelopeglycoprotein (BaEV), the murine leukemia envelope protein (MuLV), and/orthe paramyxovirus Measles envelope proteins H and F, or a fragment ofany thereof that retains the ability to bind to resting T cells and/orresting NK cells. In illustrative embodiments, the pseudotyping elementis VSV-G. As discussed elsewhere herein, the pseudotyping element caninclude a fusion with a T cell activation element, which in illustrativeembodiments, can be a fusion with any of the envelope proteinpseudotyping elements, for example MuLV or VSV-G, with an anti-CD3antibody. In further illustrative embodiments, the pseudotyping elementsinclude both a VSV-G and a fusion of an antiCD3scFv to MuLV.

In any of the aspects provided herein that include a replicationincompetent recombinant retroviral particle, in some embodiments, thereplication incompetent recombinant retroviral particle comprises on itssurface a nucleic acid encoding a domain recognized by a monoclonalantibody approved biologic.

In certain illustrative embodiments of any of the reaction mixture, use,genetically modified T cell or NK cell, or method for geneticallymodifying T cells and/or NK cells aspects and embodiments providedherein, including, but not limited to, in this Exemplary Embodimentssection, unless incompatible with, or otherwise stated, the blood cellsin the reaction mixture are blood cells that were produced by a PBMCenrichment procedure and comprise PBMCs, or the blood cells inillustrative embodiments are PBMCs. In illustrative embodiments, suchembodiments including PMBC enrichment are not combined with anembodiment where the reaction mixture includes at least 10% whole blood.Thus, in certain illustrative embodiments herein, the blood cells in areaction mixture are the PBMC cell fraction from a PBMC enrichmentprocedure to which retroviral particles are added to form the reactionmixture, and in other illustrative embodiments, the blood cells in areaction mixture are from whole blood to which retroviral particles areadded to form the reaction mixture.

Unless incompatible with, or already stated in an aspect or embodiment,for any of the methods for genetically modifying and/or transducinglymphocytes (e.g. PBMCs, or T cells and/or NK cells), or uses thatinclude such methods, or genetically modified cells produced using suchmethods, and any other method or product by process, provided herein,including but not limited to in this Exemplary Embodiments section, incertain embodiments, the replication incompetent recombinantretroviruses (i.e. replication incompetent recombinant retroviralparticles) are added from a purified composition, to the PBMCs, or Tcells and/or NK cells, or T cell, or NK cells to form the reactionmixture.

Unless incompatible with, or already stated in an aspect or embodiment,for any of the replication incompetent recombinant retrovirus(s) (i.e.replication incompetent recombinant retroviral particle(s)) aspects andembodiments herein, including but not limited to in this ExemplaryEmbodiments section, in certain embodiments, the replication incompetentrecombinant retroviruses are isolated (i.e. in an isolated form), orpurified (i.e. in a purified form), and optionally can be in acontainer, a composition, a pharmaceutical composition, or an infusioncomposition.

In another aspect provided herein is a composition comprising purifiedreplication incompetent recombinant retroviruses (i.e. replicationincompetent recombinant retroviral particles), wherein the purifiedreplication incompetent recombinant retroviruses comprise:

A. one or more pseudotyping elements on its surface, wherein the one ormore pseudotyping elements are capable of binding to a T cell and/or anNK cell and facilitating membrane fusion of the recombinant retrovirusthereto;

B. a polynucleotide comprising one or more transcriptional unitsoperatively linked to one or more promoters active in T cells and/or NKcells, wherein the one or more transcriptional units encode

-   -   i. a first engineered signaling polypeptide comprising a        lymphoproliferative element, wherein the lymphoproliferative        element promotes proliferation and/or survival of T and/or NK        cells; and    -   ii. a second engineered signaling polypeptide comprising a        chimeric antigen receptor comprising an antigen-specific        targeting region, a transmembrane domain, and an intracellular        activating domain, wherein the first or second engineered        signaling polypeptides in some embodiments is optional; and

C. an activation element on its surface, wherein the activation elementis fused to a membrane attachment sequence, and wherein the activationelement is capable of binding to a T cell and/or NK cell and is notencoded by a polynucleotide in the recombinant retrovirus.

In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the purified replication incompetentrecombinant retroviruses are in a serum-free medium. In some embodimentsof the aspect immediately above, and any other aspect or embodimentherein, the purified replication incompetent recombinant retrovirusesare frozen (i.e. in a frozen state).

In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the composition is a pharmaceuticalcomposition. In some embodiments of the aspect immediately above, andany other aspect or embodiment herein, the composition is in acontainer. In some embodiments of the aspect immediately above, and anyother aspect or embodiment herein, the container is part of a kit.

In some embodiments of the aspect immediately above, or any other aspector embodiment herein, the purified replication incompetent recombinantretroviruses are the product of a purification process comprisingconcentration, diafiltration, or both concentration and diafiltration.In some subembodiments of the embodiment immediately above, and anyother aspect or embodiment herein, the purification process comprisestangential flow filtration.

In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the purified replication incompetentrecombinant retroviruses are the product of a purification processcomprising centrifugation, polyethylene glycol (PEG) precipitation,and/or depth filtration. In some subembodiments of the embodimentimmediately above, and any other aspect or embodiment herein, thepurification process comprises depth filtration and tangential flowfiltration.

In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the purified replication incompetentrecombinant retroviruses are free of scrum components. In someembodiments of the aspect immediately above, and any other aspect orembodiment herein, the purified replication incompetent recombinantretroviruses are in a buffer. In some subembodiments of this embodiment,and any other aspect or embodiment herein, the buffer is phosphatebuffered saline. In some embodiments of the aspect immediately above,and any other aspect or embodiment herein, the composition includeslactose. In some embodiments of the aspect immediately above, and anyother aspect or embodiment herein, the purified replication incompetentrecombinant retroviruses are free of non-human animal media components.In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the purified replication incompetentrecombinant retroviruses are free of xenogeneic media components. Insome embodiments of the aspect immediately above, and any other aspector embodiment herein, the purified replication incompetent recombinantretroviruses are free of bovine serum components.

In another aspect, provided herein is a composition comprising isolatedreplication incompetent recombinant retroviruses, wherein the isolatedreplication incompetent recombinant retroviruses comprise:

A. one or more pseudotyping elements on its surface, wherein the one ormore pseudotyping elements are capable of binding to a T cell and/or anNK cell and facilitating membrane fusion of the recombinant retrovirusthereto;

B. a polynucleotide comprising one or more transcriptional unitsoperatively linked to one or more promoters active in T cells and/or NKcells, wherein the one or more transcriptional units encode

-   -   iii. a first engineered signaling polypeptide comprising a        lymphoproliferative element wherein the lymphoproliferative        element promotes proliferation and/or survival of T and/or NK        cells; and    -   iv. optionally a second engineered signaling polypeptide        comprising a chimeric antigen receptor comprising an        antigen-specific targeting region, a transmembrane domain, and        an intracellular activating domain, wherein the first or second        engineered signaling polypeptides in some embodiments is        optional; and

C. an activation element on its surface, wherein the activation elementis fused to a membrane attachment sequence, and wherein the activationelement is capable of binding to a T cell and/or NK cell and is notencoded by a polynucleotide in the recombinant retrovirus.

In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the isolated replication incompetentrecombinant retroviruses are in a serum-free medium.

In some embodiments of the aspect immediately above, and any otheraspect or embodiment herein, the isolated replication incompetentrecombinant retroviruses are free of serum components. In someembodiments of the aspect immediately above, and any other aspect orembodiment herein, he isolated replication incompetent recombinantretroviruses are in a buffer. In some subembodiments of the embodimentimmediately above, and any other aspect or embodiment herein, the bufferis phosphate buffered saline. In some embodiments of the aspectimmediately above, and any other aspect or embodiment herein, thecomposition includes lactose.

The following non-limiting examples are provided purely by way ofillustration of exemplary embodiments, and in no way limit the scope andspirit of the present disclosure. Furthermore, it is to be understoodthat any inventions disclosed or claimed herein encompass allvariations, combinations, and permutations of any one or more featuresdescribed herein. Any one or more features may be explicitly excludedfrom the claims even if the specific exclusion is not set forthexplicitly herein. It should also be understood that disclosure of areagent for use in a method is intended to be synonymous with (andprovide support for) that method involving the use of that reagent,according either to the specific methods disclosed herein, or othermethods known in the art unless one of ordinary skill in the art wouldunderstand otherwise. In addition, where the specification and/or claimsdisclose a method, any one or more of the reagents disclosed herein maybe used in the method, unless one of ordinary skill in the art wouldunderstand otherwise.

EXAMPLES Example 1. Materials and Methods for Transduction Experiments

This Example provides materials and methods used in experimentsdisclosed in subsequent Examples herein.

Recombinant Lentiviral Particle Production by Transient Transfection.

293T cells (Lenti-X™ 293T, Clontech) were adapted to suspension cultureby serial growth in Freestyle™ 293 Expression Medium (ThermoFisherScientific), named F1XT cells, and were used as the packaging cells forexperiments herein unless noted otherwise.

Where noted, a typical 4 vector packaging system included 3 packagingplasmids that encoded (i) gag/pol, (ii) rev, and (iii) a pseudotypingelement such as VSV-G. The 4^(th) vector of this packaging system is thegenomic plasmid, a third generation lentiviral expression vector(containing a deletion in the 3′ LTR leading to self-inactivation) thatencoded 1 or more genes of interest. For transfections using 4 plasmids,the total DNA used (1 μg/mL of culture volume) was a mixture of the 4plasmids at the following molar ratios: 1× gag/pol-containing plasmid,1× Rev-containing plasmid, 1× viral envelope containing plasmid (VSV-Gunless noted otherwise), and 2× genomic plasmid unless noted otherwise.Where noted, a typical 5 vector packaging system was used in which a5^(th) vector encoding, for example, a T cell activation element such asantiCD3-scFvFc-GPI, was added to the otherwise 4 vector packagingsystem. For transfections using 5 plasmids, the total DNA used (1 μg/mLof culture volume) was a mixture of the 5 plasmids at the followingmolar ratios: 1× gag/pol-containing plasmid, 1× Rev-containing plasmid,1× VSV-G containing plasmid, 2× genomic plasmid, and 1× of the 5^(th)vector unless noted otherwise.

Plasmid DNA was dissolved in 1.5 ml Gibco™ Opti-MEM™ growth media forevery 30 mL of culture containing packaging cells. Polyethylenimine(PEI) (Polysciences) (dissolved in weak acid) was diluted in 1.5 mlGibco™ Opti-MEM™ to 2 μg/mL. A 3 ml mixture of PEI and DNA was made bycombining the two prepared reagents at a ratio of 2 ug of PEI to 1 ug ofDNA. After a 5-minute room temperature incubation, the two solutionswere mixed together thoroughly, and incubated at room temperature for 20more minutes. The final volume (3 ml) was added to 30 ml of packagingcells in suspension at 1×10⁶ cells/mL in a 125 mL Erlenmeyer flask. Thecells were then incubated at 37° C. for 72 hours with rotation at 125rpm and with 8% CO₂ for transfection.

After 72 hours, the supernatants were harvested and clarified bycentrifugation at 1,200 g for 10 minutes. The clarified supernatantswere decanted to a new tube. Virus was purified from the clarifiedsupernatants by centrifugation, polyethylene glycol (PEG) precipitation,or depth filtration. For purification by centrifugation, the lentiviralparticles were precipitated by overnight centrifugation at 3,300 g, at4° C. The supernatant was discarded, and the lentiviral particle pelletswere resuspended in 1:100 of the initial volume of packaging cellculture. For purification by PEG precipitation, ¼ volume PEG was addedto the clarified supernatant and incubated overnight at 4° C. Themixture was then centrifuged at 1600 g for 1 hour (for 50 ml conicaltubes) or 1800 g for 1.5 hours (for 500 ml conical tubes). Thesupernatant was discarded, and the lentiviral particle pellets wereresuspended in 1:100 of the initial volume of packaging cell culture.For purification by depth filtration, the clarified supernatants wereconcentrated by tangential flow filtration (TFF) and benzonase digested.The virus was then purified and buffer exchanged by diafiltration intothe final formulation of freezing media (PBS with 2% lactose) andoptionally frozen for vialing.

Lentiviral particles were titered by serial dilution and analysis oftransgene expression, by transduction into 293T and/or Jurkat cells andanalysis of transgene expression by FACS or qPCR for lentiviral genomeusing Lenti-X™ qRT-PCR Titration Kit (#631235) or p24 assay ELISA kitfrom Takara (Lenti-X™ p24 Rapid Titer Kit #632200).

Genomic Plasmids Used in Examples.

The following lentiviral genomic vectors encode genes and features ofinterest as indicated:

F1-3-23 encodes a CD19 CAR comprised of an anti-CD19scFv, a CD8 stalkand transmembrane region, and an intracellular domain from CD3z followedby T2A and an eTag (aCD19:CD8:CD3z-T2A-eTag).

Additional lentiviral genomic vectors are described in specificexamples.

Example 2. Transduction Efficiency of Unstimulated PBMCs Exposed for 4Hours to Retroviral Particles Pseudotyped VSV-G or Influenza HA and NAand Optionally Copseudotyped with Envelopes Derived from VSV-G, MV, orMuLV, and Further, Optionally, Displaying an Anti-CD3 scFv on theirSurfaces

In this example, lentiviral particles pseudotyped or cospeudotyped withvarious different envelope proteins and optionally displaying a T cellactivation element were exposed to unstimulated human PBMCs for 4 hoursand transduction efficiency was assessed.

Recombinant lentiviral particles were produced in F1XT cells. The cellswere transiently transfected using PET with a genomic plasmid andseparate packaging plasmids encoding gag/pol, rev, and an envelopeplasmid. For certain samples, the transfection reaction mixture alsoincluded a plasmid encoding UCHT1scFvFc-GPI, a copseudotyping envelope,or a copseudotyping envelope fused to an antiCD3scFv. The genomicplasmid used for samples in this example was F1-0-03 as disclosed inother examples herein. The pseudotyping and copseudotyping plasmids usedfor samples in this example encoded envelope proteins from VSV-G (SEQ IDNO:336), U-VSV-G (SEQ ID NO: 455) in which the anti-CD3 scFv from UCHT1was fused to the amino terminus of the VSV-G envelope, influenza HA fromH1N1 PR8 1934 (SEQ ID NO: 311) and NA from H10N7-HKWF446C-07 (SEQ IDNO:312), U-MuLV (SEQ ID NO:341) in which the anti-CD3 scFv from UCHT1was fused to the amino terminus of the MuLV envelope, U-MuLV variants inwhich 8 to 31 C-terminal amino acids were deleted from the cytoplasmictail, U-MuLVSUx (SEQ ID NO: 454) in which the furin-mediated cleavagesite Lys-Tyr-Lys-Arg in U-MuLV was replaced with the Ile-Glu-Gly-Argpeptide, or MVHΔ24 (SEQ ID NO: 315) in which the C-terminal 24 aminoacids of the measles virus H protein were removed.

In certain samples the U-MuLV envelope protein was endcoded on the revpackaging plasmid in tandem in the format U-MuLV-IRES2-rev (MuLVIR) orin the format U-MuLV-T2A-rev (MuLV2R). By putting the copseudotypingelement on a packaging vector such as rev, 4 rather than 5 separateplasmids were used to transfect packaging cells. It was observed hereinthat transfecting with 4 rather than 5 plasmids resulted in higher viraltiters.

On Day 0, PBMCs were prepared from buffy coats from 2 donors asdescribed in Example 1 without any additional steps to remove monocytes.After isolation, 1×10⁶ unstimulated PBMCs in 1 ml of X-Vivo15 wereseeded into each well of a 96 deep-well plates. Viral particles wereadded at an MOI of 1 or 10 as indicated, and the plates were incubatedfor 4 hours at 37° C. and 5% CO₂. After the 4 hour exposure, the cellswere pelleted for 5 minutes at 400 g and washed 3 times by resuspendingthe cells in 2 mls of DPBS+2% HSA and centrifuging for 5 minutes at 400g, before the cells in each well were resuspended in 1 ml X-Vivo15 andincubated at 37° C. and 5% CO₂. No exogenous cytokines were added to thesamples at any time. Each sample was run in duplicate using PBMCs fromeach of the 2 donors. Samples were collected at Day 6 to determinetransduction efficiencies based on eTAG, and CD3 expression asdetermined by FACs analysis using a lymphocyte gate based on forward andside scatter.

FIG. 3A shows the total number of live cells per well on Day 6 followingtransduction. Compared to samples exposed to viral particles pseudotypedwith VSV-G alone, samples exposed to viral particles pseudotyped withVSV-G and also displaying UCHT1 had a greater number of cells per well.This was observed both when UCHT1scFv was displayed as a GPI-linkedscFvFc and when the scFv was fused to either the VSV-G or MuLV viralenvelopes. Not to be limited by theory, the stimulation of CD3+ T and NKcells by the antiCD3 scFv is believed to lead to proliferation andsurvival which can account for at least a portion of this increase incell number.

FIG. 3B shows the percent of CD3+ cells transduced as measured by eTAGexpression. Samples exposed to viral particles pseudotyped with VSV-Gthat also either displayed UCHT1ScFvFc-GPI or were copseudotyped withU-MuLV, U-MuLVSUx, U-VSV-G, or MVHΔ24 had higher transductionefficiencies than samples exposed to viral particles pseudotyped withVSV-G alone that didn't display an antiCD3 antibody. Among the 4 samplestested in this experiment at an MOI of 10, the efficiency by whichVSV-G+UCHT1scFvFc-GPI viral particles transduced CD3+ unstimulated PBMCswas 64.3%, 66.3%, 78.0% and 76.7%. Among the 4 samples tested in thisexperiment at an MOI of 10, the efficiency by which VSV-G+U-MuLV viralparticles transduced CD3+ unstimulated PBMCs was 37.6%, 43.8%, 20.5%,and 30.8%. When copseudotyped with VSV-G, individual variants of U-MuLVin which the 4, 8, 12, 16, 20, 24, 28, and 31 C-terminal amino acidswere deleted, transduced CD3+ unstimulated PBMCs in 4 hours similar tofull length U-MuLV (not shown). Similarly, when copseudotyped withVSV-G, individual variants of U-MuLVSUx in which the Factor X cleavagesite (AAAIEGR) between the transmembrane (TM) and surface (SU) units wasreplaced with (G4S)3 or “AAAIAGA”, transduced CD3+ unstimulated PBMCs in4 hours similar to U-MuLVSUx (not shown). Among the 4 samples tested inthis experiment at an MOI of 10, the efficiency by which VSV-G+MVHΔ24viral particles transduced CD3+ unstimulated PBMCs was 64.5%, 62.4%,72.3%, and 71.5%. In a separate experiment, viral particles pseudotypedwith influenza HA from H1N1 PR8 1934 and NA from H10N7-HKWF446C-07transduced CD3+ unstimulated PBMCs with comparable efficiency to viralparticles copseudotyped with VSV-G+U-MuLV.

Example 3. Efficient Genetic Modification of Resting Lymphocytes byExposure of Whole Blood to Recombinant Retroviral Particles for 4 HoursFollowed by a PBMC Enrichment Procedure

In this example, unstimulated human T cells and NKT cells wereeffectively genetically modified by a 4 hour incubation of a reactionmixture that included whole blood and retroviral particles that werepseudotyped with VSV-G and displayed a T cell activation element ontheir surface. PBMCs were subsequently isolated from the transductionreaction mixture using a traditional density gradientcentrifugation-based PBMC enrichment procedure. Transduction of CD3+cells was assessed by expression of the eTag transgene using flowcytometry.

Depth filtration was used to purify the following lentiviral particlesused in this Example: F1-3-23 pseudotyped with VSV-G (F1-3-23G); andF1-3-23 pseudotyped with VSV-G and displaying the T cell activationelement, UCHT1-scFvFc-GPI (F1-3-23GU).

10 ml samples of whole fresh blood in Vacutainer tubes containinganticoagulants were purchased. (StemExpress, San Diego). Theanticoagulant in individual samples was either EDTA 1.8 mg/ml orNa-Heparin 16 USP units per mL of blood. Recombinant lentiviralparticles were added directly to the Vacutainer tubes of whole blood atan MOI of 5 (assuming 1×10⁶ PBMCs/ml of blood) to initiate contacting ofthe lentiviral particles to lymphocytes in the whole blood, andincubated for 4 hours, at 37° C., 5% CO₂ with gentle mixing every hourto disrupt any sedimentation. After the 4 hour incubation, PBMCs fromeach whole blood sample were isolated individually using SepMate50 tubes(STEMCELL Technologies) according to the manufacturer's protocol. PBMCswere collected in 15 ml conical tubes and washed by resuspending thecells in 10 mls DPBS+2% HSA, and centrifuging them for 5 minutes at 400g. This wash procedure was repeated 3 times before the cells wereresuspended in 10 ml X-Vivo15 and cultured upright in T75 flasks at 37°C. and 5% CO₂. No exogenous cytokines were added to the samples at anytime. Samples were collected at Day 6 to determine transductionefficiencies based on eTag and CD3 expression on live cells asdetermined by FACs analysis using a lymphocyte gate based on forward andside scatter.

FIGS. 4A and 4B show histograms of the absolute live cell count per ml(FIG. 4A) and the percentage of CD3+eTag+ cells (i.e. transduced Tcells) (FIG. 4B) at Day 6 after transduction of whole blood. Consistentwith our previous results and the results of others studyingtransduction of isolated PBMCs, we see in this Example that recombinantretroviral particles pseudotyped with VSV-G alone are extremelyinefficient at transducing PBMCs in whole blood. We have seenpreviously, however, that recombinant retroviral particles pseudotypedwith VSV-G and displaying a T cell activation element, are capable ofefficiently transducing isolated PBMCs. Surprisingly, these histogramsshow that a PBMC enrichment step is not required for retroviralparticles to efficiently transduce PBMCs present in whole blood. Rather,retroviral particles pseudotyped with VSV-G and displayingantiCD3-scFvFc when added directly to whole blood containing ananticoagulant can effectively genetically modify and transduce PBMCstherein. Genetic modification can be achieved by a contacting andincubation that is as brief as 4 hours before the cells are washed toremove free recombinant retroviral particles. After the cells aregenetically modified, they can be effectively isolated using a PBMCenrichment procedure. As shown in this Example, the anticoagulant can beEDTA or Na-Heparin. Similar results were obtained using ACD as theanticoagulant in other experiments.

Example 4. Time Course of Retroviral Transduction of Unstimulated PBMCsby Exposure Times of 4 Hours to Less than 1 Minute

In this experiment recombinant lentiviral particles were contacted andincubated with unstimulated PBMCs for between 4 hours and less than 1minute, and were examined for their ability to transduce the PBMCs andpromote their survival and/or proliferation in vitro in the absence ofany exogenous cytokines.

Methods

Recombinant lentiviral particles were produced in 293T cells (Lenti-X™293T, Clontech) that were adapted to suspension culture in Freestyle™293 Expression Medium (Thermo Fisher Scientific). The cells weretransiently transfected using PEI with a genomic plasmid and separatepackaging plasmids encoding gag/pol, rev, and a pseudotyping plasmidencoding VSV-G as described in Example 3 of WO 2019/055946. For certainsamples, the transfection reaction mixture also included a plasmidencoding UCHT1scFvFc-GPI as further described in Example 3 of WO2019/055946. Two genomic plasmids were used in this example. The firstplasmid included a Kozak sequence, a CD8a signal peptide, a FLAG tag,and an anti-CD19:CD8:CD3z CAR followed by a triple stop sequence(F1-3-253). The second plasmid included a Kozak sequence, a CD8a signalpeptide, a FLAG tag, an anti-CD19:CD8:CD3z CAR, T2A, and the CLE DL3A-4(E013-T041-S186-S051) followed by a triple stop sequence (F1-3-451).

On Day 0, PBMCs were enriched from buffy coats (San Diego Blood Bank)from 2 donors by density gradient centrifugation with Ficoll-PaquePREMIUM® (GE Healthcare Life Sciences) and SepMate™-50 (Stemcell™Technologies) according to the manufacturer's instructions. Noadditional steps were taken to remove monocytes. After isolation, thePBMCs were diluted to 1×10⁶ PBMCs per 1 ml of X-Vivo15 (LONZA) and 1 mlwas seeded into each well of 96 deep-well plates. Cells from each donorwere also set aside for phenotype analysis by FACS. No anti-CD3,anti-CD28, IL-2, IL-7, or other exogenous cytokine was added to activateor otherwise stimulate the lymphocytes prior to transduction. Lentiviralparticles were added directly to the non-stimulated PBMCs at an MOIof 1. The transductions were incubated at 37° C. and 5% CO₂ for either 4hours, 2 hours, 30 minutes, 15 minutes, 7.5 minutes, 5 minutes, 2.5minutes or not incubated at all before the cells were spun down using a5 minute centrifugation at 400 g, and then washed 3 times in 1 ml ofDPBS+2% HSA, using 5 minute centrifugations at 400 g. Thus, for acalculation of combined transduction and incubation times. 5 minutescould be added to account for the first centrifugation, in which it isbelieved that the vast majority of lentiviral particles not associatedwith cells, were separate away from tire cells. The cells in each wellwere then resuspended in 1 ml X-Vivo15 and incubated at 37° C. and 5%CO₂. For samples treated with antiviral drugs, dapivirine ordolutegravir was added to a final concentration of 10 μM during thetransduction and the transduction reaction was incubated at 37° C. and5% CO₂ for 4 hours. The drugs were replenished at the sameconcentrations in the recovery medium after the three washes. Noexogenous cytokines were added to the samples at any time. Samples werecollected at Day 6 and transduction efficiencies based on FLAGexpression was determined by FACS analysis using a lymphocyte gate basedon forward and side scatter.

Results

In this example, an incubation period of less than 1 minute was found tobe as effective at promoting the transduction of unstimulated PBMCs byrecombinant lentiviral particles as was an incubation period of 4 hours.FIG. 5 shows the CD3+FLAG+ absolute cell count (per ul) at Day 6 aftertransduction of unstimulated PBMCs from 1 Donor by the differentrecombinant lentiviral particles for the indicated period of time. Theability of each of the recombinant lentiviral particles to transducePBMCs was similar across all incubation periods. This is particularlyevident for the lentiviral particles that express anti-CD3scFvFc-GPI andhad higher transduction efficiencies than their non anti-CD3scFvFc-GPIexpressing counterparts. For all incubation times examined, the totalnumber of transduced PBMCs was greater in those samples transduced by[F1-3-451GU] than by [F1-3-253GU] indicating that the DL3A CLE encodedin F1-3-451 is promoting the survival and/or proliferation of thesecells. The inhibition of transduction by dapivirine, a reversetranscriptase, and dolutegravir, an integrase inhibitor, as shown inFIG. 5 demonstrate that genetic modification and transgene expression bythese PBMCs is not pseudotransduction, but rather is the result oftransduction in which the viral transgene RNA is reverse transcribed,integrated into the genomes of PBMCs, and expressed. Similar resultswere observed using PBMCs from the second Donor.

Example 5. miRNA Expression Increased In Vivo Survival and/orProliferation of Transduced Cells Expressing a CAR

In this example, two miRNA libraries (Library 314 and Library 315) ofcandidate (putative) blocks of 4 miRNA precursors were assembled inseries from pools of individual miRNA precursors. The miRNA blocks wereinserted into the EF-1 alpha intron of lentiviral constructs encoding anEF-1 alpha promoter driving expression of a CAR. Human PBMCs weretransduced with lentiviral particles encoding these libraries, andinjected into tumor-bearing mice. After 20 days, the tumors wereharvested and the identity of the miRNA blocks in the PBMCs from thetumors was determined by PCR followed by Sanger Sequencing. Thus, thescreen identified miRNA blocks that are able to promote theproliferation and/or survival of transduced PBMCs in a tumor.

Methods Library Preparation

108 gBlocks® Gene Fragments were used to generate a library ofconstructs each containing 4 miRNA precursors in series in positions 1(P1), 2 (P2), 3 (P3), and 4 (P4). Each gBlock® was specific to P1, P2,P3, or P4 and contained a miR-155 framework (SEQ ID NO:457), including a5′ arm and a 3′ arm as described in Example 17 of WO 2019/055946, inwhich a unique miRNA fragment targeting an mRNA transcript correspondingto 1 of 27 different genes was used to replace the miR-155 stem-loopprecursor. For clarity, the sequences of miRNA fragments differed foreach position P1-P4 even among miRNA fragments that targeted mRNAtranscripts corresponding to the same gene. The gBlocks® for eachposition contained a unique 40 bp overlap sequence and the type IIsassembly method was used to assemble combinations of four gBlocks® intheir prescribed order, to generate the library. By these methods, atotal diversity of 531,441 unique constructs (27 miRNA at P1×27 miRNA atP2×27 miRNA at P3×27 miRNA at P4) was possible.

The library of miRNA constructs was separately cloned into the EF-1alpha intron A of F1-1-315 and F1-2-314 to generate Library 315 andLibrary 314, respectively. In addition to the EF-1 alpha promoter,F1-1-315 included a CD8a signal peptide, an anti-ROR2:CD28:CD3z CAR,T2A, and an eTag. Similarly, in addition to the EF-1 alpha promoter,F1-2-314 included a CD8a signal peptide, an anti-Axl:CD8:CD3z CAR, T2A,and an eTag. FIGS. 26A and 26B of WO 2019/055946 include a similarlentiviral vector with an EF-1 alpha promoter, including intron A with 4miRNA precursors, that drove expression of GFP instead of either CAR.

The 27 gene targets in this example and the sequence identificationnumbers for DNA sequences corresponding to the miRNAs in each positionare shown in Table 2 below.

TABLE 2 SEQ ID NOs. of DNA sequences corresponding to miRNA at eachposition for each target. Gene Target Position 1 Position 2 Position 3Position 4 cCBL SEQ ID NO: 342 SEQ ID NO: 343 SEQ ID NO: 344 SEQ ID NO:345 CD3z SEQ ID NO: 346 SEQ ID NO: 347 SEQ ID NO: 348 SEQ ID NO: 349 PD1SEQ ID NO: 350 SEQ ID NO: 351 SEQ ID NO: 352 SEQ ID NO: 353 CTLA4 SEQ IDNO: 354 SEQ ID NO: 355 SEQ ID NO: 356 SEQ ID NO: 357 TIM3 SEQ ID NO: 358SEQ ID NO: 359 SEQ ID NO: 360 SEQ ID NO: 361 LAGS SEQ ID NO: 362 SEQ IDNO: 363 SEQ ID NO: 364 SEQ ID NO: 365 SMAD2 SEQ ID NO: 366 SEQ ID NO:367 SEQ ID NO: 368 SEQ ID NO: 369 TNFRSF10B SEQ ID NO: 370 SEQ ID NO:371 SEQ ID NO: 372 SEQ ID NO: 373 PPP2CA SEQ ID NO: 374 SEQ ID NO: 375SEQ ID NO: 376 SEQ ID NO: 377 TNFRSF6 SEQ ID NO: 378 SEQ ID NO: 379 SEQID NO: 380 SEQ ID NO: 381 BTLA SEQ ID NO: 382 SEQ ID NO: 383 SEQ ID NO:384 SEQ ID NO: 385 TIGIT SEQ ID NO: 386 SEQ ID NO: 387 SEQ ID NO: 388SEQ ID NO: 389 A2AR SEQ ID NO: 390 SEQ ID NO: 391 SEQ ID NO: 392 SEQ IDNO: 393 AHR SEQ ID NO: 394 SEQ ID NO: 395 SEQ ID NO: 396 SEQ ID NO: 397EOMES SEQ ID NO: 398 SEQ ID NO: 399 SEQ ID NO: 400 SEQ ID NO: 401 SMAD3SEQ ID NO: 402 SEQ ID NO: 403 SEQ ID NO: 404 SEQ ID NO: 405 SMAD4 SEQ IDNO: 406 SEQ ID NO: 407 SEQ ID NO: 408 SEQ ID NO: 409 TGFBR2 SEQ ID NO:410 SEQ ID NO: 411 SEQ ID NO: 412 SEQ ID NO: 413 PPP2R2D SEQ ID NO: 414SEQ ID NO: 415 SEQ ID NO: 416 SEQ ID NO: 417 TNFSF6 SEQ ID NO: 418 SEQID NO: 419 SEQ ID NO: 420 SEQ ID NO: 421 CASP3 SEQ ID NO: 422 SEQ ID NO:423 SEQ ID NO: 424 SEQ ID NO: 425 SOCS2 SEQ ID NO: 426 SEQ ID NO: 427SEQ ID NO: 428 SEQ ID NO: 429 TIEG1 SEQ ID NO: 430 SEQ ID NO: 431 SEQ IDNO: 432 SEQ ID NO: 433 JunB SEQ ID NO: 434 SEQ ID NO: 435 SEQ ID NO: 436SEQ ID NO: 437 Cbx3 SEQ ID NO: 438 SEQ ID NO: 439 SEQ ID NO: 440 SEQ IDNO: 441 Tet2 SEQ ID NO: 442 SEQ ID NO: 443 SEQ ID NO: 444 SEQ ID NO: 445HK2 SEQ ID NO: 446 SEQ ID NO: 447 SEQ ID NO: 448 SEQ ID NO: 449

Lentiviral Particle Production

Library 315 and Library 314 were separately used to produce lentiviralparticles in 30 ml suspension cultures of 293T cells. The lentiviralparticles were harvested and concentrated by PEG precipitation. Otherdetails regarding lentiviral particle production are provided in Example17 of WO 2019/055946.

Transduction

On Day 0, PBMCs were isolated from ACD peripheral blood and 5.0×10⁷viable PBMCs were seeded into each of two 1L G-Rex devices in 100 mlwith Complete OpTmizer™ CTS™ T-Cell Expansion SFM supplemented with 100IU/ml IL-2 (Novoprotein, GMP-CD66), 10 ng/ml IL-7 (Novoprotein,GMP-CD47), and 50 ng/ml anti-CD3 antibody (Novoprotein, GMP-A018) toactivate the PBMCs, which included T cells and NK cells, for viraltransduction. Lentiviral particles were added directly to the activatedPBMCs in 1 G-Rex for Library 315 and the other G-Rex for Library 314 atan MOI of 5, and incubated overnight. The G-Rex devices were incubatedin a standard humidified tissue culture incubator at 37° C. and 5% CO₂with additions of 100 IU/ml recombinant human IL-2 and 10 ng/mlrecombinant human IL-7 solution every 48 hours and the cultures wereexpanded until day 12 at which time the cells are predominantly T cells.Other details regarding PBMC enrichment, transduction, and ex vivoexpansion are provided in Example 16 of WO 2019/055946.

Tumor Inoculation and Administration of Transduced Cells

A xenograft model using NOD Scid Gamma (NSG) mice was chosen to probethe ability of human PBMCs transduced with lentiviral particles ofLibrary 315 or Library 314 to survive and/or proliferate in vivo, wherethe tumors expressed or did not express the antigen recognized by theCAR encoded in the genomes of these lentiviral particles. Mice werehandled in accordance with Institutional Animal Care and Use Committeeapproved protocols. Subcutaneous (sc) tumor xenografts were establishedin the hind flank of 12 week old femaleNOD-Prkdc^(scid)Il2rg^(tml)/Begen (B-NSG) mice (Beijing Biocytogen Co.Ltd.). Briefly, cultured CHO cells, cultured CHO cells transfected tostably express human ROR2 (CHO-ROR2) or human AXL (CHO-AXL) wereseparately washed in DPBS (Thermo Fisher), counted, resuspended in coldDPBS and mixed with an appropriate volume of Matrigel ECM (Corning;final concentration 5 mg/mL) at a concentration of 0.47×10⁶ cells/200 μlon ice. Animals were prepared for injection using standard approvedanesthesia with hair removal (Nair) prior to injection. 200 μl of eithercell suspension in ECM was injected sc into the rear flanks for CHOcells (n=2), CHO-ROR2 cells (n=1), and CHO-AXL cells (n=1),respectively.

5 days after tumor inoculation, 1 mouse bearing a CHO tumor and 1 mousebearing a CHO-ROR2 tumor were dosed intravenously (IV) by tail veininjection with 200 μl DPBS containing 1×10⁷ PBMCs transduced withlentiviral particles from Library 315 after 12 days of ex vivo culture.Similarly, 5 days after tumor inoculation, 1 mouse bearing a CHO tumorand 1 mouse bearing a CHO-Axl tumor were dosed intravenously (IV) bytail vein injection with 200 μl DPBS containing 1×10⁷ PBMCs transducedwith lentiviral particles from Library 314.

Tumor Harvesting and DNA Sequencing

On day 20 after dosing with transduced PBMCs, the tumors were excised.DNA from half of each tumor was extracted and 4 ug from each tumor wasused as a template in a PCR reaction for 25 cycles to amplify theEF-1alpha intron. The amplicons were cloned into a sequencing vector,transformed into bacteria, and streaked onto plates. 18 total colonies(5 per mouse) were selected and DNA was prepared and analyzed usingSanger sequencing to determine the sequences of a sample of the miRNAconstructs present in the tumor.

Results

A mouse xenograft model was used to determine whether miRNA targetingspecific gene transcripts were able to increase the proliferation and/orsurvival of transduced PBMCs expressing CARs in vivo, where thexenografts were tumors with or without expression of the target antigenof the CARs. For this analysis, a library of miRNA constructs wasgenerated consisting of miRNAs directed against 27 distinct targets. ThemiRNA constructs analyzed contained 4 positions for 4 separate miRNAs,as shown in FIG. 26B and Example 17 and Example 18 of WO 2019/055946.Tumor DNA was analyzed by sequencing the EF-1alpha intron to identifywhich miRNA constructs were present 20 days after injection oftransduced PBMCs, and therefore which miRNA constructs increasedproliferation and/or survival.

531,441 different combinations of 4 miRNAs in series were possible. Ofthe 18 EF-1alpha introns sequenced, 13 contained a miRNA construct whereall 4 miRNA in the construct were directed against one target, and 2contained miRNA constructs directed to more than 1 target. Table 3 belowshows the miRNA species recovered from each of the 4 tumors examined inthis example.

TABLE 3 Identity of miRNA target at each position of the miRNAconstructs that were sequenced. Posi- Posi- Posi- Posi- Library Tumortion 1 tion 2 tion 3 tion 4 Library CHO FAS FAS FAS FAS 315 FAS FAS FASFAS AHR AHR AHR AHR FAS FAS FAS FAS CD3z CD3z CD3z CD3z Library CHO-ROR2NA NA NA NA 315 FAS FAS FAS FAS FAS FAS FAS FAS cCBL cCBL cCBL cCBLLibrary CHO Cbx Cbx Cbx Cbx 314 cCBL cCBL cCBL cCBL HK2 HK2 HK2 HK2 NANA NA NA Library CHO-AXL NA NA NA NA 314 FAS FAS FAS FAS FAS FASL SMAD4HK2 SMAD4 SMAD4 SMAD4 SMAD4 HOMES NA EOMES AHR

Notably, 6 EF-1 alpha introns contained a miRNA construct with all 4miRNA directed against TNFRSF6 (FAS). 2 EF-1 alpha introns contained amiRNA construct with all 4 miRNA directed against cCBL. For each of AHR,CD3z, Cbx, and HK2, 1 EF-1 alpha intron was identified that contained anmiRNA construct with all 4 miRNA directed against that gene transcript.“NA” indicated that no miRNA block was identified in that position.Together, these results indicate that knocking down transcripts encodingFAS, cCBL, CD3z, Cbx, HK2, FASL, SMAD4, EOMES, and AHR can promote thesurvival and/or proliferation of T cells in the tumor microenvironment.The identification of 4 miRNA in series to FAS under each condition in 6of the 18 samples examined indicates that knocking down FAS transcriptsconfers a particular advantage for survival and/or proliferation.Furthermore, this data suggests that there is a dosage effect such that4 species of miRNA directed to FAS, cCBL, AHR, CD3z, Cbx, and HK2, leadsto greater knockdown of transcripts encoding these genes than does 1, 2,or 3 species, and that this increased knockdown confers a survivaland/or proliferation advantage.

Example 6. Identification of Candidate Chimeric PolypeptideLymphoproliferative Elements Using an In Vivo Assay

In this example, two chimeric polypeptide libraries (Library 6 andLibrary 8) of candidate (putative) chimeric lymphoproliferative elements(CLEs) were assembled into viral vectors from pools ofextracellular-transmembrane block sequences, intracellular blocksequences, and a barcode library according to the chimericpolypeptide-encoding construct provided in FIG. 6. The chimeric librarycandidates (putative CLEs) were screened for the ability of thecandidate chimeric polypeptides to promote expansion of T cells in vivo.

Library Constructs

Two libraries were made and analyzed in this study; Library 6 andLibrary 8. The libraries shared a common structure, which is shown inFIG. 6. FIG. 6 provides a schematic of a non-limiting, exemplarytransgene expression cassette containing a polynucleotide sequenceencoding a CAR and a candidate CLE from a library having 4 modulesdriven by an EF-1 alpha promoter and a Kozak-type sequence (GCCGCCACC(SEQ ID NO:450)), in a lenti viral vector backbone. Each candidatelymphoproliferative element included 4 modules; an extracellular module(P1), a transmembrane module (P2), and 2 intracellular modules (P3 andP4). The P1 module encoded an eTAG at the 5′ terminus of a c-Jun domain.A triple stop sequence (TAATAGTGA (SEQ ID NO:451)) separated P4 from aDNA barcode (P5). A WPRE(GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCC TG (SEQID NO:452)) was present between the last stop codon (starting 4 bp afterthe last nucleotide of P5) and the 3′ LTR (which started 83 nucleotidesafter the last nucleotide of the WPRE).

The CAR and P1 were separated by a polynucleotide sequence encoding aT2A ribosomal skip sequence. The general design and construction of thelibrary, including the barcode, was as disclosed in Example 11 of WO2019/055946, except for the P1 and P2 domains, as set out in more detaillater in this Example.

The design of Library 6 and Library 8 differed only in thepolynucleotide encoding the CAR. The CAR of Library 6 encoded anMRB-ASTR that has an scFv that recognizes human A XL, a CD28 stalk andtransmembrane sequence (SEQ ID NO:25), a CD28 intracellular domaindeleted for Lck binding (ICΔ) (SEQ ID NO:55), and an intracellularactivating domain from CD3z (SEQ ID NO: 28). The CAR of Library 8encoded a FLAG-tagged MRB-ASTR that has an scFv that recognizes humanROR2, a CD8 stalk and transmembrane sequence (SEQ ID NO:24), a CD137intracellular domain (SEQ ID NO:53), and an intracellular activatingdomain from CD3z (SEQ ID NO: 28).

Synthesis of Viral Vectors and Lentiviral Production

Vectors were synthesized and lentiviral particles were produced for eachlibrary as disclosed in Example 11 of WO 2019/055946.

Transduction and Culturing of PBMCs

Whole human blood from 2 healthy donors was collected and processedseparately using a Sepax 2 S-100 device to obtain PBMCs as described inExample 12 of WO 2019/055946. 4.75e7 or 5e7 viable PBMCs for Libraries 6and 8, respectively, were seeded into each of two 1L G-Rex devices in100 ml and activated, transduced, and the cultures were expanded for 12days as described in Example 5 above. 3.9e9 total cells were recovered(82-fold expansion) for Library 6, 9.71 e7 of which were CD3+eTAG+transduced T cells. 2.47e9 total cells were recovered (49-foldexpansion) for Library 8, 2.44e8 of which were CD3+eTAG+ transduced Tcells. 4e6 cells from each expansion were set aside and frozen for lateranalysis by next generation sequencing.

Tumor Inoculation and Administration of Transduced Cells

A xenograft model using NSG mice was chosen to probe the ability ofhuman PBMCs transduced with lentiviral particles of Library 6 or Library8 to survive and/or proliferate in vivo, where the tumors expressed ordid not express the antigen recognized by the CAR encoded in the genomesof these lentiviral particles. Subcutaneous (sc) CHO, CHO-ROR2, orCHO-AXL tumor xenografts were established in the hind flanks of B-NSG(Beijing Biocytogen Co. Ltd.) mice as described in Example 5.

5 days after tumor inoculation, 6 mice bearing CHO tumors and 5 micebearing CHO-Axl tumors were dosed intravenously (IV) by tail veininjection with 200 μl DPBS containing 7×10⁷ PBMCs transduced withlentiviral particles from Library 6. Similarly, 5 days after tumorinoculation, 6 mice bearing CHO tumors and 5 mice bearing CHO-ROR2tumors were dosed IV by tail vein injection with 200 μl DPBS containing7×10⁷ PBMCs transduced with lentiviral particles from Library 8. Micebearing CHO tumors, CHO-AXL tumors, or CHO-ROR2 tumors were also dosedwith 200 μl DPBS alone as controls.

Tissue Harvesting, Isolation of Human CD45+ Cells, and DNA Sequencing

Approximately 100 μl of blood was collected from each mouse on days 7,14, and 21 (for Library 6) or days 7, 14, and 19 (for Library 8) afterdosing with transduced PBMCs. Spleen and tumor was also collected whenthe mice were euthanized on day 21 or day 19. Half of each tissue wasprocessed to isolate human CD45+ cells by mechanically disrupting thetissue, enzymatic digestion with collagenase IV and DNAse I, andmagnetic isolation of cells using hCD45 antibody (Biolegend, 304004).Genomic DNA was prepared from these hCD45+ cells and corresponds to“purified spleen” and “purified tumor” samples. Genomic DNA was prepareddirectly from the other half of each tissue and corresponds to “nonpurified spleen” and “non purified tumor. Purified genomic DNA wassequenced using an Illumina HiSeq, generating paired-end 150 bp reads.Usually, a subset of 10 million reads was extracted from each indexedfastq file and processed for analysis using barcode reader, a custom Rscript engineered to extract barcode sequences based on the presence ofa constant region. Purified genomic DNA was also sequenced on a PacBiosequencing system to obtain longer read lengths to associate barcodeswith constructs.

qPCR

Genomic DNA (gDNA) isolated from tissue samples were evaluated for thepresence of transduced lymphocytes by bioanalytical qPCR. Genomic DNAwas isolated from the samples using the QIAamp DNA Blood Mini kit(Qiagen 51106) and the DNA was further cleaned using the QIAamp DNAMicro Kit (56304). A TaqMan assay (Thermo Fisher) was performed on theisolated genomic DNA using a primer and probe set specific for the 5′LTR of lentivirus to quantitate lentivirus copy number per ug of tissue.

Data Analysis

DNA barcodes were identified in a 20 million subset of Illumina HiSeqsequenced reads. Count data for all samples was assembled and barcodespresent in less than 2 samples were considered artifactual anddiscarded. Count data from pre-injection PBMCs was used as arepresentation of the initial barcode population. Full length constructswere identified using an association table created by Long ReadSequencing of a few select samples. After summing up counts for barcodesmapping to the same construct, all data was scaled based onqPCR-quantified lentivirus copy number per ug of tissue. Samples withvery-low lentivirus copy numbers were removed from the analysis. Rankingof CAR/antigen signal-independent chimeric polypeptide candidates wasobtained by calculating the total counts for each construct in eachtissue of interest from mice bearing CHO tumors devoid of the cognatetarget antigen recognized by the CAR. Ranking for CAR/antigensignal-dependent drivers was obtained using the following formula:MR*−log 10(P), where the MR was the mean ratio between the count valuesin the mice bearing tumors with antigen (CHO-AXL or CHO-ROR2) and tumorswithout antigen (CHO) and P was the p value obtained from a one-sidedMann-Whitney-Wilcoxon test comparing the count values in the micebearing tumors with or without antigen. One-sided Mann-Whitney-Wilcoxontests were used to determine whether a particular part was enriched ascompared with all other represented parts for a specific position.Individual tissue p values were aggregated using the Stouffer sumzmethod to obtain final rankings. Full construct rankings were obtainedby averaging individual tissue ranks.

Results

In this experiment, chimeric polypeptide candidates were designed tohave 4 test domains, which included an extracellular domain (P1), atransmembrane domain (P2), a first intracellular domain (P3), and asecond intracellular domain (P4) (FIG. 6). As explained in Examples 11and 12 of WO 2019/055946, the constructs included a DNA barcode to aidin analysis and identification of the construct using next-generationsequencing. Additionally, all of the constructs included nucleic acidsequences encoding a recognition and/or elimination domain in flame withthe extracellular domain. The constructs in this Example also encoded aCAR directed to human AXL or human ROR2 upstream of the chimericpolypeptide candidate (FIG. 6). The extracellular domains (P1),transmembrane domains (P2), first intracellular domains (P3), and secondintracellular domains (P4) used to generate the chimeric polypeptidecandidates in Library 6 and Library 8 were the same as in Example 12 ofWO 2019/055946. The libraries did not include all of the possiblecombinations of P1-P4 domains.

The number of constructs present after transduction of PBMCs and 12 daysof growth in culture in the presence of exogenous cytokines wasdetermined for both Library 6 and Library 8 by counting the number ofindividual barcodes that were present in more than one read in the day12 cultured sample. Of the 697,410 potential combinations, 219,649 and127,634 different constructs were detected for Library 6 and Library 8,respectively. Detailed information about the top candidates analyzed canbe determined from Table 1 and Tables 4-8. The coding system forconstructs is the same as explained for Examples 11 and 12 of WO2019/055946.

After culturing for 12 days, transduced PBMCs were injected into micebearing tumors with or without antigen. PBMCs transduced with constructsfrom Library 6, which encoded the anti-AXL CAR, were injected into micebearing CHO tumors or CHO-AXL tumors, and PBMCs transduced withconstructs from Library 8, which encoded the anti-ROR2 CAR, wereinjected into mice bearing CHO tumors or CHO-ROR2 tumors. After 21 or 19days of in vivo expansion (Library 6 and Library 8, respectively),samples from the blood, spleen, and tumor of each mouse were harvested.Half of each spleen and tumor was processed to isolate CD45+ cells andis referred to herein in this example as a “purified” sample. DNA fromeach sample from each mouse (blood, non-purified spleen, purifiedspleen, non-purified tumor, and purified tumor) was sequenced. Thebarcodes on each construct were used to identify and sum the number ofsequencing reads for each construct in each sample.

A non-parametric analysis was used to identify constructs that promotedPBMC cell proliferation in vivo in either a CAR/antigensignal-independent or CAR/antigen signal-dependent manner. To identifychimeric polypeptide candidates that were CAR/antigensignal-independent, each sample of each construct was ranked based onthe number of sequencing reads in mice bearing CHO tumors. The topconstructs were identified as having the best average rank of the 5tissue samples. The top 100 chimeric polypeptide candidates that wereCAR/antigen signal-independent for Library 6 and Library 8 are shown inTables 31 and 32, respectively.

To identify chimeric polypeptide candidates that were CAR/antigensignal-dependent, the tanking for each sample included the ratio ofreads between mice bearing tumors with antigen (CHO-AXL or CHO-ROR2) andmice bearing tumors without antigen (CHO). The top 100 chimericpolypeptide candidates that were CAR/antigen signal-dependent forLibrary 6 and Library 8 are shown in Tables 33 and 34, respectively.

An additional analysis was run to identify noteworthy chimericpolypeptide candidates that were CAR/antigen signal-independent. Forthis analysis, 20 parts were first identified that performed the bestfor any P2, P3, or P4 position, based on a statistical test to determinewhether a particular part was enriched as compared with all otherrepresented parts for a specific position. In this combined analysis,from constructs that included at least one of these 20 parts,best-performing constructs from either Library 6 or Library 8 wereidentified based on the sum of the normalized counts in mice bearing CHOtumors. The 30 best-performing chimeric polypeptide candidates accordingto this analysis that were CAR/antigen signal-independent are shown inTable 8.

Several of the CLEs identified in the library screen and shown in Table8 were generated as individual chimeric polypeptides in lentivirusconstructs behind the anti-AXL CAR as configured in Library 6 and run inconfirmatory in vitro screens. Frozen PBMCs from 3 donors were thawedand rested in Complete OpTmizer™ CTS™ T-Cell Expansion SFM supplementedwith 100 IU/ml of IL-2 and 10 ng/ml IL-7 overnight in a standardhumidified tissue culture incubator at 37° C. and 5% CO₂. The PBMCs wereactivated on Day 0 with 50 ng/ml anti-CD3 and transduced on Day 1 withviral particles at an MOI of 5. On Day 2 the PBMCs were transferred tothe wells of a 24-well G-Rex plate and cultured in Complete OpTmizer™CTS™ T-Cell Expansion SFM in the absence of any exogenous cytokinesuntil Day 35 days. In replicate experiments performed using PBMCs from 3donors, CLE's with P2, P3, and P4 configurations T001-S121-S212 andT044-S186-S053 showed particularly noteworthy expansion on Days 14, 21,28, and 35.

The disclosed embodiments, examples and experiments are not intended tolimit the scope of the disclosure or to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. It should be understood that variations in the methods asdescribed may be made without changing the fundamental aspects that theexperiments are meant to illustrate.

Those skilled in the art can devise many modifications and otherembodiments within the scope and spirit of the present disclosure.Indeed, variations in the materials, methods, drawings, experiments,examples, and embodiments described may be made by skilled artisanswithout changing the fundamental aspects of the present disclosure. Anyof the disclosed embodiments can be used in combination with any otherdisclosed embodiment.

In some instances, some concepts have been described with reference tospecific embodiments. However, one of ordinary skill in the artappreciates that various modifications and changes can be made withoutdeparting from the scope of the invention as set forth in the claimsbelow. Accordingly, the specification and figures are to be regarded inan illustrative rather than a restrictive sense, and all suchmodifications are intended to be included within the scope of invention.

TABLE 1 Parts, names, and amino acid sequences for domains oflymphoproliferative parts P1-P2, P1, P2, P3, and P4. Part NameAmino Acid Sequence M001 eTAG IL7RA InsMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSPPCL (interleukinFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG7 receptor)LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPEINNSSGEMDPILLPPCLTISILSFFSVALLVILACVL (SEQ ID NO: 84) M002 eTAG IL7RA InsMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSPPCLFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG(interleukin 7LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQPEINNSSGEMDPILreceptor) LPPCLTISILSFFSVALLVILACVL (SEQ ID NO: 85) M007 Myc Tag LMP1MEQKLISEEDLEHDLERGPPGPRRPPRGPPLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFANC_007605_1LMLIIIILIIFIFRRDLLCPLGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 86)M008 Myc LMP1MEQKLISEEDLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPNC_007605_1LGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 87) M009 LMP1MEHDLERGPPGPRRPPRGPPLSSSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIINC_007605_1FIFRRDLLCPLGALCILLLMITLLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 88) M010 LMP1MSLGLALLLLLLALLFWLYIVMSDWTGGALLVLYSFALMLIIIILIIFIFRRDLLCPLGALCILLLMITNC_007605_1LLLIALWNLHGQALFLGIVLFIFGCLLVLGIWIYLLEMLWRLGATIWQLLAFFLAFFLDLILLIIALYLQQNWWTLLVDLLWLLLFLAILIWM (SEQ ID NO: 89) M012 eTAG CRLF2MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG1 NM_022148_3LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATCQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGAETPTPPKPKLSKCILISSLAILLMVSLLLLSLW (SEQ ID NO: 90) M013 eTAG CRLF2MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG1 NM_022148_3LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQAETPTPPKPKLSKCILISSLAILLMVSLLLLSLW (SEQ ID NO: 91) M018 eTAG CSF2RBMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_000395_2FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFLSAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTESVLPMWVLAIEIFLTIAVLLAL (SEQ ID NO: 92) M019 eTAG CSF2RBMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_000395_2FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTESVLPMWVLALIEIFLTIAVLLAL (SEQ ID NO: 93) M024 eTAG CSF3RMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEIRGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG1 NM_000760_3LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTPEGSELHIILGLFGLLLLLNCLCGTAWLCC (SEQ ID NO: 94) M025 eTAG CSF3RMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG1 NM_000760_3LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTPEGSELHIILGLFGLLLLLNCLCGTAWLCC (SEQ ID NO: 95) M030 eTAG EPORMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQGSLAVVSLNITSLG1 NM_000121_3LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYKIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTPSDLDPCCLTLSLILVVILVLLTVLALLS (SEQ ID NO: 96) M031 eTAG EPORMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG1 NM_000121_3LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTPSDLDPCCLTLSILVVILVLLTVLALLS (SEQ ID NO: 97) M036 eTAG GHRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSSL1 NM_000163_4GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHEPCLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGTLPQMSQFTCCEDFYFPWLLCIIFGIFGLTVMLFVFLFS (SEQ ID NO: 98) M037 eTAG GHRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDStranscript variantFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSSL1 NM_000163_4GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQTLPQMSQFTCCEDFYFPWLLCIIFGIFGLTVMLFVFLFS (SEQ ID NO: 99) M042 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSafter Fn F523CFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSGLIL27RARSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPENM_004843_3PRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECKIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGHLPDNTLRWKVLPGILCLWGLFLLGCGLSLA (SEQ ID NO: 100) M043 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSafter Fn F523CFTHTPPDLPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQGSLAVVSLNITSLGIL27RALRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQHLPDNTLRWKVLPGNM_004843_3 ILCLWGLFLLGCGLSLA (SEQ ID NO: 101) M048 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSafter Fn S505NFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGMPL NM_005373_2LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGETATETAWISLVTALHLVLGLNAVLGLLLL (SEQ ID NO: 102) M049 eTAG truncatedMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSafter Fn S505NFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGMPL NM_005373_2GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQETATETAWISLVTALHLVLGLNAVLGGGG (SEQ ID NO: 103) E006 eTag 0A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_002228_3FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTRGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKV (SEQ ID NO: 104) E007 eTag 01 JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_002228_3FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVA (SEQ ID NO: 105) E008 eTag 2A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_002228_3FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQMANITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAA (SEQ ID NO: 106) E009 eTag 3A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_002228_3FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFLSAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNCSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAAA (SEQ ID NO: 107) E101 eTag 4A JUNMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSNM_002228_3FTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFLSAVVSLNITSLGLRLSKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCPGEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVAAAA (SEQ ID NO: 108) E011 Myc Tag 0A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVNM_002228_3 (SEQ ID NO: 109) E012 Myc Tag 1A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVNM_002228_3 A (SEQ ID NO: 110) E013 Myc Tag 2A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVNM_002228_3 AA (SEQ ID NO: 111) E014 Myc Tag 3A JUNMTILGTTFGMVFSLLQVVSGEQKLISEEDLLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVNM_002228_3 AAA (SEQ ID NO: 112) E015 Myc Tag 4A JUNMTILGTTFGMVFSLLQVVSGEQLKLISEELLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVNM_002228_3 AAAA (SEQ ID NO: 113) T001 CD2 transcriptLIIGICGGGSLLMVFVALLVFYI (SEQ ID NO: 114) variant 1 NM_001328609_1 T002CD3D transcript GIIVTDVIATLLLALGVFCFA (SEQ ID NO: 115) variant 1NM_000732_4 T003 CD3E VMSVATIVIDICITGGLLLLVYYWS (SEQ ID NO: 116)NM_000733_3 T004 CD3G GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 117) NM_000073_2T005 CD3Z CD247 LCYLLDGILFIYGVILTALFL (SEQ ID NO: 118)transcript variant 1 NM_198053_2 T006 CD4 transcriptMALIVLGGVAGLLFIGFIGLGIFF (SEQ ID NO: 119) variant 1 and 2 NM_000616_4T007 CD8A transcript IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 120) variant 1NM_001768_1 T008 CD88 transcriptLGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 121) variant 2 NM_172213_3 T009 CD27ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 122) NM_001242_4 T010 CD28 transcriptFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 123) variant 1 NM_006139_3 T011CD40 transcript ALVVIPIIFGILFAILLVLVFI (SEQ ID NO: 124) variant 1 and 6NM_001250_5 T012 CD79A transcriptIITAEFIILLFCAVVPGTLLLF (SEQ ID NO: 125) variant 1 NM_001783_3 T013CD79B transcript GIIMIQTLLIIFIIVPIFLLL (SEQ ID NO: 126) variant 3NM_001039933_2 T014 CRLF2 transcriptFILISSLAILLMVSLLLLSLW (SEQ ID NO: 127) variant 1 NM_022148_3 T015CRLF2 transcript CILISSAILLMVSLLLLSLW (SEQ ID NO: 128) variant 1NM_022148_3 T016 CSF2RA transcriptNLGSVYIYVLLLIVGTLVCGIVLGFLF (SEQ ID NO: 129) variant 7 and 8NM_00111529_1 T017 CSF2RB MWVLALIVIFLTIAVLLAL (SEQ ID NO: 130)NM_000395_2 T018 CSF2RB MWVLALIEFLTIAVLLAL (SEQ ID NO: 131) NM_000395_2T019 CSF3R transcript IILGLFGLLLLLTCLCGTAWLCC (SEQ ID NO: 132) variant 1NM_000760_3 T020 CSF3R transcriptIILGLFGLLLLNCLCGTAWLCC (SEQ ID NO: 133) variant 1 NM_000760_3 T021EPOR transcript LILTLSLILVVILVLLTVLALLS (SEQ ID NO: 134) variant 1NM_000121_3 T022 EPOR transcriptCCLTLSLILVVILVLLTVLALLS (SEQ ID NO: 135) variant 1 NM_000121_3 T023FCER1G LCYILDAILFLYGIVLTLLYC (SEQ ID NO: 136) NM_004106_1 T024 FCGR2CIIVAVVTGIAVAAIVAAVVALIY (SEQ ID NO: 137) NM_201563_5 T025FCGRA2 transcript IIVAVVIATAVAAIVAAVVALIY (SEQ ID NO: 138) variant 1NM_001136219_1 T026 GHR transcriptFPWLLIIFGIFGLTVMLFVFLFS (SEQ ID NO: 139) variant 1 NM_000163_4 T027GHR transcript FPWLLCIIFGIFGLTVMLFVFLFS (SEQ ID NO: 140) variant 1NM_000163_4 T028 ICOS FWLPIGCAAFVVVCILGCILI (SEQ ID NO: 141) NM_012092.3T029 IFNAR1 IWLIVGICIALFALPFVIYAA (SEQ ID NO: 142) NM_000629_2 T030IFNAR2 transcript IGGIITVFLIALVLTSTIVTL (SEQ ID NO: 143) variant 1NM_207585_2 T031 IFNGR1 SLWIPVVAALLLFLVLSLVFI (SEQ ID NO: 144)NM_000416_2 T032 IFNGR2 transcriptVILISVGTFSLLSVLAGACFF (SEQ ID NO: 145) variant 1 NM_001329128_1 T033IFNLR1 FLVLPSLLILLLVIAAGGVIW (SEQ ID NO: 146) NM_170743_3 T034IL1R1 transcript HMIGICVTLTVIIVCSVFIYKIF (SEQ ID NO: 147) variant 2NM_001288706_1 T035 IL1RAP transcriptVLLVVILIVVYHVYWLEMVLF (SEQ ID NO: 148) variant 1 NM_002182_3 T036IL1RL1 transcript IYCIIAVCSVFLMLINVLVII (SEQ ID NO: 149) variant 1NM_016232.4 T037 IL1RL2 AYLIGGLIALVAVAVSVVYIY (SEQ ID NO: 150)NM_003854.2 T038 IL2RA transcript VAVAGCVFLLISVLLLSGL (SEQ ID NO: 151)variant 1 NM_000417_2 T039 IL2RB transcriptIPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 152) variant 1 NM_000878_4 T040IL2RG VVISVGSMGLIISLLCVYFWL (SEQ ID NO: 153) NM_000206_2 T041IL3RA transcript TSLLIALGTLLALVCVFVIC (SEQ ID NO: 154) variant 1 and 2NM_002183_3 T042 IL4R transcriptLLLGVSVSCIVILAVCLLCYVSIT (SEQ ID NO: 155) variant 1 NM_000418_3 T043IL5RA transcript FVIVIMATICFILLILSLIC (SEQ ID NO: 156) variant 1NM_000564_4 T044 IL6R transcript TFLVAGGLSAFGTLLCIAIVL (SEQ ID NO: 157)variant 1 NM_000565_3 T045 IL6ST transcriptAIVVPVCLAFLLTTLLGVLFCF (SEQ ID NO: 158) variant 1 and 3 NM_002184_3 T046IL7RA ILLTISILSFFSVALLVILACVL (SEQ ID NO: 159) NM_002185_3 T047IL7RA Ins PPCL ILLPPCLTISILSFFSVALLVILACVL (SEQ ID NO: 160)(interleukin 7 receptor) T048 IL9R transcriptGNTLVAVSIFLLLTGPTYLLF (SEQ ID NO: 161) variant 1 NM_002186_2 T049IL10RA transcript VIIFFAFVLLLSGALAYCLAL (SEQ ID NO: 162) variant 1NM_001558_3 T050 IL10RB WMVAVILMASVFMVCLALLGCF (SEQ ID NO: 163)NM_000628_4 T051 IL11RA SLGILSFLGLVAGALALGLWL (SEQ ID NO: 164)NM_001142784_2 T052 IL12RB1 transcriptWLIFFASLGSFLSILLVGVLGYLGL (SEQ ID NO: 165) variant 1 and 4 NM_005535_2T053 IL12RB2 transcript WMAFVAPSICIALLMVGIFST (SEQ ID NO: 166)variant 1 and 3 NM_001559_2 T054 IL13RA1LYITMLLIVPVIVAGAIIVLLLYL (SEQ ID NO: 167) NM_001560_2 T055 IL13RA1FWLPFGFILILVIFVTGLLL (SEQ ID NO: 168) NM_000640_2 T056 IL15RA transcriptVAISTSTVLLCGLSAVSLLACYL (SEQ ID NO: 169) variant 4 NM_001256765_1 T057IL17RA VYWFITGISILLVGSVILLIV (SEQ ID NO: 170) NM_014339_6 T058 IL17RBLLLLSLLVATWVLVAGIYLMW (SEQ ID NO: 171) NM_018725_3 T059IL17RC transcript WALVWLACLLFAAALSLILLL (SEQ ID NO: 172) variant 1NM_153460_3 T060 IL17RD transcriptAVAITVPLVVISAFATLFTVM (SEQ ID NO: 173) variant 2 NM_017563_4 T061IL17RE transcript LGLLILALLALLTLLGVVLAL (SEQ ID NO: 174) variant 1NM_153480_1 T062 IL18R1 transcriptGMIIAVLILVAVVCLVTVCVI (SEQ ID NO: 175) variant 1 NM_003855_3 T063IL18RAP GVVLLYILLGTIGTLVAVLAA (SEQ ID NO: 176) NM_003853_3 T064IL20RA transcript IIFWYVLPISITVFLFSVMGY (SEQ ID NO: 177) variant 1NM_014432_3 T065 IL20RB VLALFAFVGFMLILVVVPLFV (SEQ ID NO: 178)NM_144717_3 T066 IL21R transcript GWNPHLLLLLLLVIVFIPAFW (SEQ ID NO: 179)variant 2 NM_181078_2 T067 IL22RA1YSFSGAFLFSMGFLVAVLCYL (SEQ ID NO: 180) NM_021258_3 T068 IL23RLLLGMIVFAVMLSILSLIGIF (SEQ ID NO: 181) NM_144701_2 T069 IL27RAVLPGILFLWGLFLLGCGLSLA (SEQ ID NO: 182) NM_004843_3 T070 IL27RAVLPGILCLWGLFLLGCGLSLA (SEQ ID NO: 183) NM_004843_3 T071IL31RA transcript IILITSLIGGGLLILIILTVAYGL (SEQ ID NO: 184) variant 1NM_139017_5 T072 LEPR transcriptAGLYVIVPVIISSSILLLGTLLI (SEQ ID NO: 185) variant 1 NM_002303_5 T073 LIFRVGLIIAILIPVAVAVIVGVVTSILC (SEQ ID NO: 186) NM_001127671_1 T074 MPLISLVTALHLVLGLSAVLGLLLL (SEQ ID NO: 187) NM_005373_2 T075 MPLISLVTALHLVLGLNAVLGLLLL (SEQ ID NO: 188) NM_005373_2 T076 OSMR transcriptLIHILLPMVFCVLLIMVMCYL (SEQ ID NO: 189) variant 4 NM_001323505_1 T077PRLR transcript TTVWISVAVLSAVICLIIVWAVAL (SEQ ID NO: 190) variant 1NM_000949_6 T078 TNFRSF4 VAAILGLGLVLGLLGPLAILL (SEQ ID NO: 191)NM_003327_3 T079 TNFRSF8 PVLDAGPVLFWVILVWVVVVGSSAFLLC (SEQ ID NO: 192)transcript variant 1 NM_001243_4 T080 TNFRSF9IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 193) NM_001561_5 T081 TNFRSF14WWFLSGSLVIVIVCSTVGLII (SEQ ID NO: 194) transcript variant 1 NM_003820_3T082 TNFRSF18 LGWLTVVLLAVAACVLLLTSA (SEQ ID NO: 195) transcript variant1 NM_004195_2 S036 CD2 transcriptTKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRvariant 1VQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 196)NM_001328609_1 S037 CD3D transcriptGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 197) variant 1NM_000732_4 S038 CD3EKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI NM_000733_3(SEQ ID NO: 198) S039 CD3GGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 199)NM_000073_2 S042 CD4 transcriptCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI (SEQ ID NO: 200)variant 1 and 2 NM_000616_4 S043 CD8A transcriptLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 201) variant 1 NM_001768_6S044 CD88 transcriptRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNPWILKT (SEQ ID NO: 202)variant 2 NM_172213_3 S045 CD88 transcriptRRRRARLRFMKQLRLHPLEKCSRMDY (SEQ ID NO: 203) variant 3 NM_172101_3 S046CD88 transcript RRRRARLRFMKQFYK (SEQ ID NO: 204) variant 5 NM_004931_4S047 CD27QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 205)NM_001242_4 S048 mutated Delta LckRSKRSRLLHSDYMNMTPRRPGPTRKHYQAYAAARDFAAYRS (SEQ ID NO: 206)CD28 transcript variant 1 NM_006139_3 S049 CD28 transcriptRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 207) variant 1NM_006139_3 S050 CD40 transcriptKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETHGCQPVTQEDGKESRISVQERQvariant 1 and 6 (SEQ ID NO: 208) NM_001250_5 S051 CD79A transcriptSESSEKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQvariant 5 (SEQ ID NO: 209) NM_001322421_1 S052 CD79A transcriptRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP variant 1(SEQ ID NO: 210) NM_001783_3 S053 CD79B transcriptLDKDDSKAGMEEDHTYEGLDIDQTATEDIVTLRTGEVKWSVGEHPGQE (SEQ ID NO: 211)variant 3 NM_001039933_2 S054 CRLF2 transcriptKLWRVKKFLIPSVPDPKSIFPGLFEIHQGNFQEWITDTQNVAHLHKMAGAEQESGPEEPLVVQLAKTEAvariant 1ESPRMLDPQTEEKEASGGSLQLPHQPLQGGDVVTIGGFTFVMNDRSYVAL (SEQ ID NO: 212)NM_022148_3 S057 CSFR2BRFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFNM_000395_2GDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEFLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC (SEQ ID NO: 213) S058 CSF2RA transcriptKRFLRIQRLFPPVPQIKDKLNDNHEVEDEIIWEEFTPEEGKGYREEVLTVKEIT variant 7 and 8(SEQ ID NO: 214) NM_001161529_1 S059 CSF2RA transcriptKRFLRIQRLFPPVPQIDKDLNDNHEVEDEMGPQRHHRCGWNLYPTPGPSPGSGSSPRLGSESSLvariant 9 (SEQ ID NO: 215) NM_001161531_1 S062 CSF3R transcriptSPNRKNPLWPSVPDPAHSSLGSWVPTIMEEDAFQLPGLGTPPITKLTVLEEDEKKPVPWESHNSSETCGvariant 1LPTLVQTYVLQGDPRAVSTQPQSQSGTSDQVLYGQLLGSPTSPGPGHYSRCDSTQPLLAGLTPSPKSYENM_000760_3NLWFQASPLGTLVTPAPSQEDDCVFGPLLNFPLLQGIRVHGMEALGSF (SEQ ID NO: 216) S063CSF3R transcriptSPNRKNPLWPSVPDPAHSSLGSWVPTIMEELPGPRQGQWLGQTSEMSRALTPHPCVQDAFQLPGLGTPPvariant 3ITKLTVLEEDEKKPVPWESHNSSETCGLPTLVQTYVLQGDPRAVSTQPQSQSGTSDQVLYGQLLGSPTSNM_156039_3PGPGHYLRCDSTQPLLAGLTPSPKSYENLWFQASPLGTLVTPAPSQEDDCVFGPLLNFPLLQGIRVHGMEALGSF (SEQ ID NO: 217) S064 CSF3R transcriptSPNRKNPLWPSVPDPAHSSLGSWVPTIMEEDAFQLPGLGTPPITKLTVLEEDEKKPVPWESHNSSETCGvariant 4LPTLVQTYVLQGDPRAVSTQPQSQSGTSDQAGPPRRSAYFKDQIMLHPAPPNGLLCLFPITSVLNM_172313_2 (SEQ ID NO: 218) S069 EPOR transcriptHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMvariant 1QAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALANM_000121_3SKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKYLYLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS (SEQ ID NO: 219) S072 EPOR transcriptHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMvariant 1QAVEPGTDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPGGSVDIVAMDEGSEASSCSSALANM_000121_3SKPSPEGASAASFEYTILDPSSQLLRPWTLCPELPPTPPHLKFLFLVVSDSGISTDYSSGDSQGAQGGLSDGPYSNPYENSLIPAAEPLPPSYVACS (SEQ ID NO: 220) S074 FCER1GRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 221) NM_004106_1S075 FCGR2CCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQPEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNM_201563_5 NDHVNSNN (SEQ ID NO: 222) S076 FCGRA2 transcriptCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPvariant 1 NDHVNSNN (SEQ ID NO: 223) NM_001136219_1 S077 GHR transcriptKQQRIKMLILPPVPVPKIKGIDPDLLKEGKLEEVNTILAIHDSYKPEFHSDDSWVEFIELDIDEPDEKTvariant 1EESDTDRLLSSDHEKSHSNLGVKDGDSGRTSCCEPDILETDFNANDIHEGTSEVAQPQRLKGEADLLCLNM_000163_4DQKNQNNSPYHDACPATQQPSVIQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKNKAGMSQCDMHPEMVSLCQENFLMDNAYFCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTGEHVPGSEMPVPDYTSIHIVQSPQGLILNATALPLPDKEFLSSCGYVSTDQLNKIMP (SEQ ID NO: 224) S080 ICOSVWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 225) NM_012092.3 S081IFNAR1KVFLRCINYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKVFIIENISTIATVEETNQTDEDHKKNM_000629_2 YSSQTSQDSGNYSNEDESESKTSEELQQDFV (SEQ ID NO: 226) S082IFNAR2 transcriptKWIGYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSvariant 1GGGYTMHGLTVRPLGQASATSTESQLIDPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPCERRKSPLNM_207585_2QDPFPEEDYSSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPTFPSPSSEGLWSEDAPSDQSDTSESDVDLGDGYIMR (SEQ ID NO: 227) S083IFNAR2 transcriptKWIGYICLRNSLPKVLRQGLAKGWNAVAIHRCSHNALQSETPELKQSSCLSFPSSWDYKRASLCPSDvariant 2 (SEQ ID NO: 228) NM_000874_4 S084 IFNGR1CFYIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSLEKEVVCEEPLSPATVPGMHNM_000416_2TEDNPGKVEHTEELSSITEVVTTEENIPDVVPGSHLTPIERESSSPLSSNQSEPGSIALNSYHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFGYDKPHVLVDLLVDDSGKESLIGYRPTEDSKEFS (SEQ ID NO: 229) S085 INFNGR2 transcriptLVLKYRGLIKYWHFTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPEKEQEDVLQTLvariant 1 (SEQ ID NO: 230) NM_001329128_1 S086 IFNLR1KTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTRGVRPTPRVRAPATQQTRWNM_170743_3KKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR(SEQ ID NO: 231) S087 IFNLR1 transcriptKTLMGNPWFQRAMMPRALELTRGVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIEPPvariant 2SFLGQEHQAPGHSEAGGVDSGRPRAPLVPSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPNM_173064_2GGDGHQESLPPPEFSKDSGFLEELPEDNLSSWATWGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 232) S098IL1R1 transcriptKIDIVLWYRDSCYDFLPIKVLPEVLEKQCGYKLFIYGRDDYVGEDIVEVINENVKKSRRLIIILVRETSvariant 2GFSWLGGSSEEQIAMYNALVQDGIKVVLLELEKIQDYEKMPESIKFIKQKHGAIRWSGDFTQGPQSAKTNM_001288706_1RFWKNVRYHMPVQRRSPSSKHQLLSPATKEKLQREAHVPLG (SEQ ID NO: 233) S099IL1R1 transcriptKIDIVLWYRDSCYDFLPIKASDGKTYDAYILYPKTVGEGSTSDCDIFVFKVLPEVLEKQCGYKLFIYGRvariant 3DDYVGEDIVEVINENVKKSRRLIILVRETSGFSWLGGSSEEQAIMYNALVQDGIKVVLLELEKIQDYEKNM_001320978_1MPESIKFIKQKHGAIRWSGDFTQGPQSAKTRFWKNVRYHMPVQRRSPSSKHQALLSPATKEKLQREAHVPLG (SEQ ID NO: 234) S100 IL1RAP transcriptYRAHFGTDETILDGKEYDIYVSYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFvariant 1IQKSRRLLVVLSPNYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKETKVKELKRAKTVLTVIKWNM_002182_3KGEKSKYPQGRFWKQLQVAMPVKKSPRRSSSDEQGLSYSSLKNV (SEQ ID NO: 235) S101IL1RAP transcriptYRAHFGTDETILGDKEYDIYVSYARNAEEEEFVLLTLRVLENEFGYKLCIFDRDSLPGGNTVEAFDFIQvariant 6RSRRMIVVLSPDYVTEKSISMLEFKGVMCQNSIATKLIVVEYRPLEHPHPGILQLKESVSFVSWGKEKSNM_001167931_1KHSGSKFWKALRLALPLRSLSASSGWNESCSSQSDISLDHVQRRRSRLEKPPELQSSERAAGSPPAPGTMSKHRGKSSATCRCCVTYCEGENHLRNKSRAEIHNQPQWETHLCKPVPQESETQWIQNGTRLEPPAPQISALALHHFTDLSNNNDFYIL (SEQ ID NO: 236) S102 IL1RL1 transcriptLDMFWIEATLLWRDIAKPYKTRNDGKLYDAYVVPRNYKSSTDGASRVEHFVHQILPDVLENKCGYTLCIvariant 1YGRDMLPGEDVVTAVETNIRKSRRHIFILTPQITHNKEFAYEQEVALHCALIQNDAKVILIEMEALSELNM_016232_4DMLQAEQLQDSLQHLMKVQGTIKWREDHIANKRSLNSKFWKHVRYQMPVPSKIPRKASSLTPLAAQKQ(SEQ ID NO: 237) S103 IL1RL2NIKFIDIVLWYRSAFHSTETIVDGKLYDAYVLPKPHKESQRHAVDALVLNILPEVLERQCGYKLFIFGRNM_003854.2DEFPGQAVANVIDENVKLCRRLIVIVVPESLGRGLLKNLSEEQIAVYSALIQDGMKVILIELEKIEDYTVMPESIQYIKQKHGAIRWHGDFTEQSQCMKTKFWKTVRYHNPPRRCRPFPPVQLLQHTPCYRTAGPELGSRRKKCTLTTG (SEQ ID NO: 280) S104 IL2RA transcriptTWQRRQRKSRRTI (SEQ ID NO: 239) variant 1 NM_000417_2 S105IL2RB transcriptNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTvariant 1QLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSNM_000878_4SPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRANLNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 240) S106 IL2RGERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNNM_000206_2 QHSPYWAPPCYTLKPET (SEQ ID NO: 241) S109 IL3RA transcriptRRYLVMQRLFPRIPHMKDPIGDSFQNDKLVVWEAGKAGLEECLVTEVQVVQKT variant 1 and 2(SEQ ID NO: 242) NM_002183_3 S110 IL4R transcriptKIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHvariant 1KAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDNM_000418_3FQEGREGIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS (SEQ ID NO: 243) S113 IL4R transcriptKIKKEWWDQIPNPARSRLVAIIIQDAQGSQEEKRSRGQEPAKVPHWKNCLTKLLPCFLEHNMKRDEDPHvariant 1KAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDNM_000418_3FQEGREGIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNLVQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVFSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS (SEQ ID NO: 244) S115 IL5RA transcriptKICHLWIKLFPPIPAPKSNIKDLFVTTNYEKAGSSETEIEVICYIEKPGVETLEDSVF variant 1(SEQ ID NO: 245) NM_000564_4 S116 IL6R transcriptRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDARDPRSvariant 1 PYDISNTDYFFPR (SEQ ID NO: 246) NM_000565_3 S117IL6ST transcriptNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSvariant 1 and 3LDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRNM_002184_3SESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ (SEQ ID NO: 247) S120 IL7RA Isoform 1WKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLNM_002185.4EESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ(SEQ ID NO: 248) S121 IL7RA Isoform 3WKKRIKPIVWPSLPDHKKTLEHLCKKPRKVSVFGA (SEQ ID NO: 249) (C-term deletion)(interleukin 7 receptor) S126 IL9R transcriptKLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTvariant 1CGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLAYLPQEDWAPTSLTRPAPPDSEGSNM_002186_2RSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPALACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF (SEQ ID NO: 250) S129 IL10RA transcriptQLYVRRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEFAFLKVSPELKNLDLHGSTDSGFGSTKvariant 1PSLQTEEPQFLLPDPHPQADRTLGNREPPVLDGDSCSSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNM_001558_3NSRGQGGSGIDLVQNSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAAVAFQGYLRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQSSE (SEQ ID NO: 251) S130IL10RBALLWCVYKKTKYAFSPRNSLPQHLKEFLGHPHHNTLLFFSFPLSDENDVFDKLSVIAEDSESGKQNPGDNM_00628_4 SCSLGTPPGQGPQS (SEQ ID NO: 252) S135 IL11RARLRRGGKDGSPKPGFLASVIPVDRRPGAPNL (SEQ ID NO: 253) NM_001142784_2 S136IL12RB1 transcriptNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEvariant 1 and 4 GAPELALDTELSLEDGDRCKAKM (SEQ ID NO: 254) NM_005535_2S137 IL12RB1 transcriptNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEvariant 3 GAPELALDTELSLEDGDRCDR (SEQ ID NO: 255) NM_001290023_1 S138IL12RB2 transcriptHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHvariant 1 and 3QVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYMKVLESRGSDPKPENM_001559_2NPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (SEQ ID NO: 256) S141 IL13RA1KRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQTKEETDSVVLEINLKKASQ NM_001560_2(SEQ ID NO: 257) S142 IL13RA2 RKPNTYPKMIPEFFCDT (SEQ ID NO: 258)NM_000640_2 S143 IL15RA transcriptKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 259) variant 4NM_001256765_1 S144 IL17RACMTWRLAGPGSEKYSDDTKYTDGLPAADLIPPPLKPRKVWIIYSADHPLYVDVVLKFAWFLLTACGTEVNM_014339_6ALDLLEEQAISEAGVMTWVGRQKQEMVESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACFGTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVYFRIQDLEMFQPGRMHRVGELSGDNYLRSPGGRQLRAALDRFRDWQVRCPDWFECENLYSADDQDAPSLDEEVFEEPLLPPGTGIVKRAPLVREPGSQACLAIDPLVGEEGGAAVAKLEPHLQPRGQPAPQPLHTLVLAAEEGALVAAVEPGPLADGAAVRLALAGEGEACPLLGSPGAGRNSVLFLPVDPEDSPLGSSTPMASPDLLPEDVREHLEGLMLSLFEQSLSCQAQGGCSRPAMVLDTPHYTPYEEEQRQSVQSDQGYISRSSPQPPEGLTEMEEEEEEEQDPGKPALPLSPEDLESLRSLQRQEEFRQLQKNSGWDTMGSESEGPSA (SEQ ID NO: 260) S145 IL17RBRHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCRSEVILEKWQKKKIAEMGPVQWLNM_018725_3ATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQDLFPLAFNLFCSDLRSQIHLHKYVVVYFREIDTKDDYNALSVCPKYHLMKDATAFCAELLHVKQQVSAGKRSQACHDGCCSL (SEQ ID NO: 261)S146 IL17RC transcriptKKDHAKGWLRLLKQDVRSGAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQvariant 1GPVAWFHAQRRQTLQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPNM_153460_3GSYVGACFDRLLHPDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT (SEQ ID NO: 262) S147 IL17RC transcriptKKDHAKAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPVAWFHAQRRQTvariant 4LQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYVGACFDRLLHNM_001203263_1PDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT (SEQ ID NO: 263) S148 IL17RD transcriptCRKKQQENIYSHLDEESSESSTYTAALPRERLRPRPKVFLCYSSKDGQNHMNVVQCFAYFLQDFCGCEVvariant 2ALDLWEDFSLCREGQREWVIQKIHESQFIIVVCSKGMKYFVDKKNYKHKGGGRGSGKGELFLVAVSAIANM_017563_4EKLRQAKQSSSAALSKFIAVYFDYSCEGDVPGILDLSTKYRLMDNLPQLCSHLHSRDHGLQEPGQHTRQGSRRNYFRSKSGRSLYVAICNMHQFIDEEPDWFEKQFVPFHPPPLRYREPVLEKFDSGLVLNDVMCKPGPESDFCLKVEAAVLGATGPADSQHESQHGGLDQDGEARPALDGSAALQPLLHTVKAGSPSDMPRDSGIYDSSVPSSELSLPLMEGLSTDQTETSSLTESVSSSSGLGEEEPPALPSKLLSSGSCKADLGCRSYTDELHAVAPL (SEQ ID NO: 264) S149 IL17RE transcriptTCRRPQSGPGPARPVLLLHAADSEAQRRLVGALAELLRAALGGGRDVIVDLWEGRHVARVGPLPWLWAAvariant 1RTRVAREQGTVLLLWSGADLRPVSGPDPRAAPLLALLHAAPRPLLLLAYFSRLCAKGKIPPPLRALPRYNM_153480_1 RLLRDLPRLLRALDARPFAEATSWGRLGARQRRQSRLELCSRLEREAARLADLG(SEQ ID NO: 265) S154 IL18R1 transcriptYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVvariant 1VPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSNM_003855_3 LKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES(SEQ ID NO: 266) S155 IL18RAPSALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYNM_003853_3GYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW (SEQ ID NO: 267) S156 IL20RA transcriptSIYRYIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDVSSLNvariant 1DPQPSGNLRPPQEEEEVKHLGYASHLMEIFCDSEENTEGTSLTQQESLSRTIPPDKTVIEYEDVRTTDINM_014432_3CAGPEEQELSLQEEVSTQGTLLESQAALAVLGPQTLQYSYTPQLQDLDPLAQEHTDSEEGPEEEPSTTLVDWDPQTGRLCIPSLSSFDQDSEGCEPSEGDGLGEEGLLSRLYEEPAPDRPPGENETYLMQFMEEWGLYVQMEN (SEQ ID NO: 268) S157 IL20RBWKMGRLLQYSCCPVVVLPDTLKITNSPQKLISCRREEVDACATAVMSPEELLRAWIS NM_144717_3(SEQ ID NO: 269) S158 IL21R transcriptSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPvariant 2RSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPNM_181078_2CSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (SEQ ID NO: 270) S161 IL22RA1SYRYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGPSSLAQPVQYSQIRVSGPREPAGAPQRNM_021258_3HSLSEITYLGQPDISILQPSNVPPPQILSPLSYAPNAAPEVGPPSYAPQVTPEAQFPFYAPQAISKVQPSSYAPQATPDSWPPSYGVCMEGSGKDSPTGTLSSPKHLRPKGQLQKEPPAGSCMLGGLSLQEVTSLAMEESQEAKSLHQPLGICTDRTSDPNVLHSGEEGTPQYLKGQLPLLSSVQIEGHPMSLPLQPPSRPCSPSDQGPSPWGLLESLVCPKDEAKSPAPETSDLEQPTELDSLFRGLALTVQWES (SEQ ID NO: 271) S165IL23RNRSFRTGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHNM_144701_2KPTDYKKENTGPLETRDYPQNSLFDNTTVVY8IPDLNTGYKPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHFNRISLLEK (SEQ ID NO: 272) S168IL27RATSGRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATNM_004843_3 APLDSGYEKHFLPTPEEGLLGPPRPQVLA (SEQ ID NO: 273) S169 IL27RATSWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATAPLDSGYEKHFLPNM_004843_3 TPEEGLLGPPRPQVLA (SEQ ID NO: 274) S170 IL31RA transcriptKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKLVVNFvariant 1GNVLQEIFTDEARTGQENNLGGEKNGYVTCPFRPDCPLGKSFEELPVSPEIPPRKSQYLRSRMPEGTRPNM_139017_5EAKEQLLFSGQSLVPDHLCEEGAPNPYLKNSVTAREFLVSEKLPEHTKGEV (SEQ ID NO: 275)S171 IL31RA transcriptKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRIKLPCSTPSDKLVIDKLVVNFvariant 4 GNVLQEIFTDEARTGQENNLGGEKNGTRILSSCPTSI (SEQ ID NO: 276)NM_001242638_1 S174 LEPR transcriptSHQRMKKLFWEDVPNPKNCSWAQGLNFQKPETFEHLFIKHTASVTCGPLLLEPETISEDISVDTSWKNKvariant 1DEMMPTTVVSLLSTTDLEKGSVCISDQFNSVNFSEAEGTEVTYEDESQRQPFVKYATLISNSKPSETGENM_002303_5EQGLINSSVTKCFSSKNSPLKDSFSNSSWEIEAQAFFILSDQHPNIISPHLTFSEGLDELLKLEGNFPEENNDKKSIYYLGVTSIKKRESGVLLTDKSRVSCPFPAPCLFTDIRVLQDSCSHFVENNINLGTSSKKTFASYMPQFQTCSTQTHKIMENKMCDLTV (SEQ ID NO: 277) S175 LEPR transcriptSHQRMKKLFWEDVPNPKNCSWAQGLNFQKMLEGSMFVKSHHHSLISSTQGHKHCGRPQGPLHRKTRDLCvariant 2 SLVYLLTLPPLLSYDPAKSPSVRNTQE (SEQ ID NO: 278) NM_001003680_3S176 LEPR transcript SHQRMKKLFWEDVPNPKNCSWAQGLNFQKRTDIL (SEQ ID NO: 279)variant 3 NM_001003679_3 S177 LEPR transcriptSHQRMKKLFWEDVPNPKNCSWAQGLNFQKKMPGTKELLGGGWLT (SEQ ID NO: 280) variant 5NM_001198688_1 S180 LIFRYRKREWIKETFYPDIPNPENCKALQFQKSVCEGSSALKTLEMNPCTPNNVEVLETRSAFPKIEDTEIISNM_001127671_1PVAERPEDRSDAEPENHVVVSYCPPIIEEEIPNPAADEAGGTAQVIYIDVQSMYQPQAKPEEEQENDPVGGAGYKPQMHLPINSTVEDIAAEEDLDKTAGYRPQANVNTWNLVSPDSPRSIDSNSEIVSFGSPCSINSRQFLIPPKDEDSPKSNGGGWSFTNFFQNKPND (SEQ ID NO: 281) S183 LMP1YYHGQRHSDEHHHDDSLPHPQQATDDSGHESDSNSNEGRHHLLVSGAGDGPPLCSQNLGAPGGGPDNGPNC_007605_1QDPDNTDDNGPQDPDNTDDNGPHDPLPQDPDNTDDNGPQDPDNTDDNGPHDPLPHSPSDSAGNDGGPPQLTEEVENKGGDQGPPLMTDGGGGHSHDSGHGGGDPHLPTLLLGSSGSGGDDDDPHGPVQLSYYD(SEQ ID NO: 282) S186 MPLRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLEILPKSSERTPLPLNM_005373_1 CSSQAQMDYRRLQPSCLGTMPLSVCPPMAESGSCCTTHIANHSYLPLSYWQQP(SEQ ID NO: 283) S189 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 1PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVNM_001172567_1PRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRLARRPRGGCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 284) S190 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 2PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVNM_002468_4PRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 285) S191 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 3PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGHMPERFDAFICYCPSDIQFVQEMIRQLENM_001172568_1QTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 286) S192MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 4PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVNM_001172569_1PRTAELAGITTLDDPLGAAGWWWLSLMITCRARNVTSRPNLHSASLQVPIRSD (SEQ ID NO: 287)S193 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 5PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGAAGWWWLSLMITCRARNVTSRPNLHSASNM_001172566_1 LQVPIRSD (SEQ ID NO: 288) S194 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRLQETQADvariant 1PRGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVNM_001172567_1 PRTAELAGITTLDDPLGHMPERFDAFICYCPSDI (SEQ ID NO: 289) S195MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 3 PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGHMPERFDAFICYCPSDINM_001172568_1 (SEQ ID NO: 290) S196 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQELTQADvariant 1PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVNM_001172567_1PRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRLARRPRGGCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRPIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 291) S197 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADvariant 2PTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVNM_002468_4PRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRIPIIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 292) S198 MYD88 transcriptMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPvariant 3TGRLLDAWQGRPGASVGRLLELLTKGRDDVLLELGPSIGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNM_001172568_1NYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRPIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 293) S199OSMR transcriptKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLIIMNVSDCDIPAIEVVSKPEGTKIQFLGTRKSLTETvariant 4ELTKPNYLYLLPTEKNHSGPGPCICFENLTYNQAASDSGSCGHVPVSPKAPSMLGLMTSPENVLKALEKNM_001323505_1NYMNSLGEIPAGETSLNYVSQLASPMFGDKDSLPTNPVEAPHCSEYKMQMAVSLRLALPPPTENSSLSSITLLDPGEHYC (SEQ ID NO: 294) S202 PRLR transcriptKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGCQDFPPTSDYEDLLVEYLEVDDSEDQHLMSvariant 1VHSKEHPSQGMKPTYLDPDTDSGRGSCDSPSLLSEKCEEPQANPSTFYDPEVIEKPENPETTHTWDPQCNM_000949_6ISMEGKIPYFHAGGSKCSTWPLPQPSQHNPRSSYHNITDVCELAVGPAGAPATLLNEAGKDALKSSQTIKSREEGKATQQREVESFHSETDQDTPWLLPQEKTPFGSAKPLDYVEIHKVNKDGALSLLPKQRENSGKPKKPGTPENNKEYAKVSGVMDNNILVLVPDPHAKNVACFEESAKEAPPSLEQNQAEKALANFTATSSKCRLQLGGLDYLDPACFTHSFH (SEQ ID NO: 295) S211 TNFRSF4ALYLLRRDQRLPPDAHKPPGGGSFTRPIQEEQADAHSTLAKI (SEQ ID NO: 296) NM_003327_3212 TNFRSF8HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGtranscript variantAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAE1 NM_001243_4PELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 297) S213TNFRSF9 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 298)NM_001561_5 S214 TNFRSF14CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNHtranscript variant (SEQ ID NO: 299) 1 NM_002830_3 S215 TNFRSF18QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWVtranscript variant (SEQ ID NO: 300) 1 NM_004195_2 S216 TNFRSF18QLGLHIWQLRKTQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 301)transcript variant 3_NM_148902_1 X001 LinkerGSGGSEGGGSEGGAATAGSGSGS (SEQ ID NO: 302)

TABLE 4 Constructs present in Library 6 Top 100 in vivo, antigenindependent. Ranking Block Sequence 1 E006-T030-S129-S047 2E006-T023-S171-S211 3 E008-T001-S121-S212 4 E006-T064-S190-S080 5E006-T047-S141-S050 6 E008-T001-S064-S047 7 E006-T048-X001-S211 8E009-T073-S062-S053 9 E010-T035-S190-S047 10 E010-T055-S192-S051 11E006-T071-S165-S076 12 E009-T075-S165-S050 13 E010-T027-S117-S053 14E007-T054-S197-S212 15 E007-T056-S170-S050 16 E007-T050-S190-S051 17E008-T060-S190-S074 18 E007-T080-S059-S080 19 E007-T057-S059-S075 20E006-T045-S177-S216 21 E010-T077-S058-S053 22 E006-T073-S120-S048 23E009-T063-S192-S053 24 E008-T067-S190-S074 25 E009-T057-S117-S074 26E007-T045-S190-S211 27 E009-T068-S083-S212 28 E007-T039-S197-S080 29E010-T036-S058-S048 30 E008-T056-S190-S050 31 E010-T026-S120-S038 32E006-T017-S062-S039 33 E009-T073-S142-X002 34 E009-T077-S192-S212 35E007-T052-S199-S049 36 E007-T061-S186-S211 37 E009-T009-S197-S038 38E008-T029-S161-S216 39 E010-T006-S190-X002 40 E010-T081-S190-X002 41E008-T045-S062-S211 42 E008-T049-S116-S076 43 E009-T029-S190-S211 44E008-T068-S158-S076 45 E007-T058-S194-S037 46 E010-T024-S115-S039 47E010-T070-S190-S216 48 E010-T049-S115-S074 49 E006-T059-S190-S051 50E006-T035-S197-S039 51 E009-T076-S190-S051 52 E007-T032-S117-S051 53E010-T078-S197-S051 54 E006-T026-S165-S037 55 E007-T081-S194-S047 56E010-T003-S194-S215 57 E009-T069-S194-S050 58 E010-T057-S190-S211 59E008-T006-S129-S216 60 E008-T078-S190-S211 61 E006-T065-S194-S080 62E009-T012-S171-S074 63 E009-T041-S165-S038 64 E006-T057-S194-S038 65E006-T012-S176-S076 66 E008-T052-S197-S050 67 E007-T016-S135-S212 68E009-T007-S192-S051 69 E006-T065-S165-S047 70 E008-T011-S135-S075 71E007-T002-S190-S051 72 E006-T037-S165-S053 73 E007-T021-S130-S212 74E010-T071-S194-S211 75 E007-T023-S158-S080 76 E008-T078-S177-S215 77E010-T008-S196-S049 78 E006-T026-S199-S053 79 E006-T027-S084-S037 80E007-T034-S189-S212 81 E010-T074-S130-S212 82 E008-T072-S192-S075 83E008-T021-S109-S039 84 E006-T065-S135-S214 85 E006-T073-X001-S074 86E008-T032-S175-X002 87 E010-T072-S192-S050 88 E008-T067-S189-S050 89E008-T073-S192-S074 90 E006-T023-S183-S076 91 E010-T041-S147-S076 92E010-T067-S130-S074 93 E008-T023-S194-S212 94 E006-T063-S190-S211 95E006-T053-S194-X002 96 E008-T019-S194-S211 97 E007-T020-S109-S050 98E006-T024-S194-S074 99 E009-T049-S194-S050 100 E008-T027-S126-S053

TABLE 5 Constructs present in Library 8 Top 100 in vivo, antigenindependent. Ranking Block Sequence 1 E006-T032-S197-S075 2E006-T013-S196-S048 3 E008-T030-S057-S037 4 E006-T069-S177-S080 5E009-T056-S104-S080 6 E006-T006-S171-S215 7 E006-T023-S117-S080 8E006-T057-S180-S051 9 E007-T032-S064-S052 10 E006-T044-S186-S053 11E009-T020-S121-S037 12 E009-T012-S154-X002 13 E010-T042-S194-S050 14E009-T062-S190-S074 15 E006-T018-S141-S213 16 E009-T026-S100-S047 17E006-T053-S186-S074 18 E010-T021-S197-S049 19 E007-T005-S143-S211 20E009-T005-S157-S216 21 E006-T038-S192-S039 22 E007-T005-S170-S076 23E009-T069-S143-S049 24 E006-T057-S189-S038 25 E008-T065-S069-S053 26E009-T042-S058-S074 27 E006-T045-S072-S051 28 E010-T011-S121-S038 29E009-T072-S154-X002 30 E010-T072-S194-S047 31 E008-T038-S165-S052 32E010-T057-S141-S050 33 E006-T056-S196-S212 34 E010-T066-S197-S051 35E008-T031-S083-S212 36 E009-T006-S062-S053 37 E010-T043-S186-S075 38E008-T003-S138-S039 39 E008-T057-S141-S049 40 E008-T056-S192-S039 41E009-T049-S199-S037 42 E006-T045-S197-S053 43 E007-T012-S130-S052 44E007-T015-S069-S038 45 E009-T065-S062-X002 46 E008-T014-X001-S051 47E008-T026-S058-S050 48 E008-T048-S161-S050 49 E006-T067-S145-S052 50E009-T049-S135-S052 51 E006-T080-S121-S074 52 E009-T044-S130-S037 53E007-T016-S165-S037 54 E008-T047-S194-X002 55 E006-T050-S186-S039 56E008-T055-X001-S216 57 E008-T013-S197-S216 58 E010-T072-S192-S212 59E007-T001-S064-S215 60 E007-T065-S197-S075 61 E010-T040-S189-S047 62E009-T039-S117-S074 63 E007-T042-S177-S048 64 E010-T061-S175-S213 65E008-T063-S069-S075 66 E008-T070-S165-S212 67 E009-T012-S064-S211 68E006-T006-S194-S211 69 E010-T035-S121-S214 70 E006-T011-S170-S211 71E006-T048-S058-S053 72 E009-T040-S058-S214 73 E009-T019-S146-S050 74E010-T045-S135-S075 75 E006-T071-S058-S049 76 E008-T031-S170-S211 77E007-T030-S176-S048 78 E008-T007-S192-S213 79 E006-T035-S121-S075 80E008-T060-S064-S214 81 E010-T077-S117-S037 82 E007-T066-S054-X002 83E008-T023-S194-S214 84 E009-T044-S083-S038 85 E007-T077-S062-S074 86E006-T063-S130-S052 87 E009-T010-S170-S074 88 E010-T072-S192-S038 89E010-T016-S168-S037 90 E010-T036-S197-S074 91 E010-T004-S194-S216 92E009-T049-S085-S075 93 E009-T059-S193-S039 94 E007-T042-S099-S053 95E008-T031-S104-S076 96 E006-T039-S115-S080 97 E006-T073-S117-S053 98E010-T032-X001-S049 99 E007-T029-S104-S049 100 E006-T072-S158-S047

TABLE 6 Constructs present in Library 6 Top 100 in vivo, antigendependent Ranking Block Sequence 1 E006-T066-S109-X002 2E010-T012-S192-S214 3 E009-T028-S130-S212 4 E010-T032-S186-S050 5E007-T052-S197-S075 6 E007-T052-S102-S049 7 E009-T023-S190-S050 8E008-T008-S194-S215 9 E010-T058-S121-S080 10 E009-T019-S194-S049 11E008-T004-S142-S212 12 E007-T012-S054-S076 13 E010-T077-S192-S074 14E006-T073-X001-S074 15 E006-T070-S197-S037 16 E006-T069-S197-S053 17E006-T061-S190-S080 18 E008-T032-S190-S213 19 E008-T022-S109-S052 20E009-T078-S190-S047 21 E009-T015-S083-S053 22 E010-T072-S146-S047 23E010-T078-S197-S051 24 E007-T063-S196-S050 25 E010-T055-S192-S051 26E006-T059-S190-S051 27 E006-T026-S199-S053 28 E010-T002-S194-S050 29E009-T075-S165-S050 30 E010-T082-X001-S052 31 E008-T032-S083-S074 32E007-T040-S192-S049 33 E007-T045-S192-S051 34 E010-T025-S194-S047 35E006-T078-S082-S048 36 E010-T082-S186-S047 37 E010-T072-S192-S050 38E007-T039-S197-S080 39 E010-T072-S186-S037 40 E008-T035-S176-S038 41E008-T056-S190-S050 42 E007-T021-S130-S212 43 E009-T049-S194-S050 44E007-T032-S117-S051 45 E009-T052-S102-S049 46 E010-T005-S192-S214 47E007-T061-S186-S211 48 E009-T057-S117-S074 49 E007-T016-S135-S212 50E009-T073-S062-S053 51 E008-T049-S116-S076 52 E006-T023-S171-S211 53E006-T048-X001-S211 54 E010-T035-S190-S047 55 E007-T054-S197-S212 56E006-T045-S177-S216 57 E006-T071-S165-S076 58 E009-T049-S194-S051 59E009-T049-S197-S051 60 E010-T072-S176-S074 61 E009-T049-S190-S051 62E008-T067-S197-S074 63 E009-T049-S196-S051 64 E006-T026-S165-S037 65E009-T049-S189-S051 66 E006-T035-S197-S039 67 E006-T012-S176-S076 68E006-T065-S194-S080 69 E009-T073-S142-X002 70 E009-T069-S194-S050 71E007-T045-S190-S211 72 E009-T076-S190-S051 73 E010-T070-S190-S216 74E010-T049-S115-S074 75 E010-T024-S115-S039 76 E007-T081-S194-S047 77E007-T058-S194-S037 78 E009-T029-S190-S211 79 E006-T073-S120-S048 80E008-T045-S062-S211 81 E009-T063-S192-S053 82 E008-T067-S190-S074 83E010-T026-S120-S038 84 E009-T068-S083-S212 85 E010-T081-S190-X002 86E008-T060-S190-S074 87 E007-T080-S059-S080 88 E010-T077-S058-S053 89E006-T047-S141-S050 90 E009-T009-S197-S038 91 E008-T001-S121-S212 92E007-T056-S170-S050 93 E008-T068-S158-S076 94 E006-T064-S190-S080 95E007-T050-S190-S051 96 E006-T030-S129-S047 97 E008-T001-S064-S047 98E007-T052-S199-S049 99 E010-T027-S117-S053 100 E010-T036-S058-S048

TABLE 7 Constructs present in Library 8 Top 100 in vivo, antigendependent Ranking Block Sequence 1 E006-T077-S129-X002 2E006-T031-S109-S216 3 E007-T057-S195-S213 4 E006-T006-S062-S052 5E008-T033-S197-S216 6 E009-T010-S177-S037 7 E006-T049-S109-S074 8E007-T029-S069-S076 9 E006-T044-S062-S053 10 E007-T048-S186-S053 11E009-T032-X001-S211 12 E010-T018-S165-S051 13 E006-T038-S154-X002 14E007-T021-S194-S211 15 E009-T005-S142-S076 16 E008-T012-S157-S216 17E009-T005-S197-S051 18 E007-T021-S190-S047 19 E010-T066-S129-S039 20E010-T033-S149-S215 21 E006-T070-S085-S076 22 E009-T041-S190-S214 23E007-T031-S130-S047 24 E008-T073-S165-X002 25 E010-T068-S194-S050 26E008-T006-S197-S213 27 E010-T072-S104-S215 28 E008-T045-S165-S080 29E008-T041-S104-S048 30 E008-T001-S165-S048 31 E009-T046-S155-S038 32E006-T026-S146-S212 33 E010-T002-S192-S039 34 E007-T052-S135-S074 35E006-T001-S158-S215 36 E008-T031-S117-S215 37 E007-T082-S142-S211 38E008-T044-X001-S211 39 E007-T029-S197-S038 40 E010-T032-X001-S049 41E009-T070-S161-X002 42 E008-T011-S135-S213 43 E007-T009-S059-S076 44E007-T037-S141-S216 45 E010-T072-S192-S038 46 E006-T015-S085-X002 47E008-T012-S146-S052 48 E008-T068-S165-S050 49 E006-T044-S192-S038 50E006-T026-S135-S074 51 E007-T042-S169-X002 52 E006-T007-S192-S049 53E008-T025-S121-S076 54 E008-T065-S192-S213 55 E008-T073-S069-S080 56E008-T073-S192-S214 57 E010-T026-S064-S074 58 E007-T001-S197-S216 59E009-T001-S109-S212 60 E007-T063-S192-S047 61 E009-T031-S063-S215 62E006-T044-S186-S053 63 E008-T040-S069-S050 64 E006-T005-S064-S213 65E007-T063-S069-S074 66 E009-T078-S192-S214 67 E007-T004-S194-S047 68E006-T057-S180-S051 69 E009-T012-S154-X002 70 E008-T073-S069-S076 71E010-T073-S189-S038 72 E009-T073-S062-S211 73 E009-T049-S142-S038 74E009-T078-S165-S074 75 E009-T078-S197-S080 76 E010-T044-S104-S048 77E009-T013-S175-S211 78 E007-T029-S197-S211 79 E006-T038-S192-S039 80E006-T048-S115-S216 81 E010-T043-S117-S048 82 E007-T012-S142-S211 83E010-T065-S130-S075 84 E007-T016-S106-S037 85 E006-T032-S138-S053 86E007-T022-S121-S076 87 E007-T070-S054-S074 88 E010-T051-S115-S051 89E010-T079-S072-S039 90 E007-T003-S142-S080 91 E009-T008-S062-S037 92E007-T063-S142-S075 93 E007-T024-S135-S074 94 E010-T057-S197-S211 95E009-T065-S145-S051 96 E008-T012-S141-S213 97 E007-T025-S202-S214 98E009-T036-S138-S047 99 E009-T032-S141-S213 100 E009-T058-S195-S048

TABLE 8 Constructs present in combined Library 6 and Library 8 sum ofmeans analysis, Top 30 in vivo, antigen independent Rank Block Sum 1E006-T006-S171-S215 370,424 2 E008-T001-S121-S212 320,942 3E009-T056-S104-S080 169,035 4 E008-T030-S057-S037 167,467 5E006-T023-S117-S080 139,222 6 E006-T032-S197-S075 120,909 7E009-T062-S190-S074 97,498 8 E007-T032-S064-S052 93,519 9E010-T072-S192-S212 84,725 10 E006-T044-S186-S053 71,737 11E006-T064-S190-S080 69,102 12 E009-T006-S062-S053 53,397 13E008-T003-S138-S039 52,634 14 E006-T038-S192-S039 49,701 15E009-T073-S062-S053 40,515 16 E009-T032-S170-S074 35,245 17E010-T021-S197-S049 33,588 18 E007-T005-S170-S076 22,931 19E007-T054-S197-S212 22,916 20 E007-T039-S197-S080 19,845 21E008-T038-S165-S052 17,583 22 E008-T078-S190-S211 16,857 23E008-T031-S083-S212 16,809 24 E010-T066-S197-S051 16,457 25E006-T056-S196-S212 15,881 26 E008-T065-S069-S053 15,512 27E008-T001-S064-S047 15,240 28 E009-T010-S170-S074 14,526 29E006-T006-S194-S211 13,077 30 E006-T045-S072-S051 12,177

What is claimed is:
 1. A method for genetically modifying T cells and/orNK cells, comprising: contacting the T cells and/or NK cells ex vivowith replication incompetent recombinant retroviruses in a reactionmixture, wherein the replication incompetent recombinant retrovirusescomprise: A. a pseudotyping element on their surface that is capable ofbinding to a T cell and facilitating membrane fusion of the replicationincompetent recombinant retrovirus thereto; B. an activation element onits surface, wherein the activation element is fused to a membraneattachment sequence, and wherein the activation element is capable ofbinding to a T cell and/or NK cell and is not encoded by apolynucleotide in the replication incompetent recombinant retrovirus;and C. a polynucleotide comprising one or more transcriptional unitsoperatively linked to one or more promoters active in the T cell,wherein the one or more transcriptional units encode a first engineeredsignaling polypeptide comprising a lymphoproliferative element, whereinthe lymphoproliferative element promotes proliferation and/or survivalof T cells, wherein the contacting is less than 12 hours, before the Tcells and/or NK cells are washed out of the reaction mixture, andwherein said contacting facilitates fusion of the replicationincompetent recombinant retroviruses to the T cells and/or NK cells toproduce the genetically modified T cells and/or NK cells.
 2. The methodof claim 1, wherein the one or more transcriptional units furtherencodes a second engineered signaling polypeptide comprising a chimericantigen receptor comprising an antigen-specific targeting region, atransmembrane domain, and an intracellular activating domain.
 3. Themethod of claim 1, wherein the first engineered signaling polypeptide isencoded in reverse orientation with respect to a viral LTR, or an activefragment thereof, in the genome of the recombinant retrovirus.
 4. Themethod of claim 1, wherein at least one of the one or more promoters isnot active in a packaging line used to produce the replicationincompetent recombinant retrovirus.
 5. The method of claim 1, wherein atleast one of the promoters is the EF1a promoter, the MSCV promoter, orthe CD3z promoter.
 6. The method of claim 1, wherein the polynucleotidefurther comprises an intron.
 7. The method of claim 6, wherein theintron encodes an shRNA or one or more miRNAs.
 8. The method of claim 6,wherein the intron is an EF1a intron.
 9. The method of claim 1, whereina first transcriptional unit of the one or more transcriptional unitsfurther comprises an intron, and wherein at least one of the one or morepromoters, the intron, and nucleic acids encoding the first engineeredsignaling polypeptide of the first transcriptional unit, are in reverseorientation with respect to a viral LTR, or an active fragment thereof,in the genome of the recombinant retrovirus.
 10. The method of claim 1,wherein the lymphoproliferative element activates a Stat3 pathway, aStat4 pathway, or a Stat5 pathway.
 11. The method of claim 1, whereinthe lymphoproliferative element comprises an intracellular signalingdomain from BTLA, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A, CD8B, CD27,mutated Delta Lck CD28, CD28, CD40, CD79A, CD79B, CRLF2, CSF2RB, CSF2RA,CSF3R, DAP10/CD28, DAP12, EPOR, FCER1G, FCGR2A, FCGR2C, FCGRA2, GHR,ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1,IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA,IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2,IL15RA, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA,IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL,MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, TNFRSF18, orZAP70.
 12. The method of claim 1, wherein the lymphoproliferativeelement is constitutively active.
 13. The method of claim 1, wherein thelymphoproliferative element is an intracellular signaling domain fromCD2, CD4, CD8A, CD8B, CD40, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR,FCGR2C, FCGR2A, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1,IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R,IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RA, IL17RB,IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1,IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR, or PRLR.
 14. The method ofclaim 1, wherein the wherein the lymphoproliferative element comprisesan intracellular signaling domain from CSF2RB, CSF3R, IFNGR1, IL2RB,IL2RG, IL6ST, IL10RA, IL17RE, IL18R1, IL22RA1, IL31RA, MPL, MyD88, OSMR,or PRLR.
 15. The method of claim 1, wherein tire lymphoproliferativeelement is a fusion with a recognition domain of a monoclonal antibodyapproved biologic.
 16. The method of claim 1, wherein expression of thelymphoproliferative element is regulated by an in vivo control element.17. The method of claim 1, wherein the activation element comprises: A.a polypeptide capable of binding to CD3; and/or B. a polypeptide capableof binding CD28.
 18. The method of claim 17, wherein the polypeptidecapable of binding to CD3 is a polypeptide capable of binding to CD3that is fused to a heterologous GPI anchor attachment sequence and thepolypeptide capable of binding to CD28 is a polypeptide capable ofbinding to CD28 that is fused to a heterologous GPI anchor attachmentsequence.
 19. The method of claim 17, wherein the polypeptide capable ofbinding to CD3 is an anti-CD3 antibody.
 20. The method of claim 19,wherein the anti-CD3 antibody is a GPI-anchored anti-CD3scFvFc.
 21. Themethod of claim 1, wherein the pseudotyping element comprises avesicular stomatitis virus (VSV-G) envelope protein, a feline endogenousvirus (RD114) envelope protein, a Measles Virus F polypeptide, or aMeasles Virus H polypeptide, or a functional fragment thereof.
 22. Themethod of claim 1, wherein the replication incompetent recombinantretrovirus is a lentivirus or a gammaretrovirus
 23. The method of claim1, wherein at least one of the one or more pseudotyping elements is theactivation element.
 24. The method of claim 1, wherein said contactingis either an initial contacting step only without any further incubatingof the replication incompetent recombinant retroviruses with the T cellsand/or NK cells in the reaction mixture, or occurs for between 30seconds and 8 hours, before the T cells and/or NK cells are washed outof the reaction mixture.
 25. The method of claim 1, wherein said one ormore transcriptional units further encode at least one inhibitory RNAmolecule that targets an mRNA encoding AHR, Cbx3, HK2, SMAD4, or HOMES.26. The method of claim 25, wherein the inhibitory RNA moleculecomprises at least one of the sequences of SEQ ID NOs: 394-401, 406-409,438-441, or 446-449.
 27. The method of claim 1, wherein the replicationincompetent recombinant retroviruses are added from a purifiedcomposition, to the T cells and/or NK cells to form the reactionmixture.
 28. A composition comprising purified replication incompetentrecombinant retroviruses, wherein the purified replication incompetentrecombinant retroviruses comprise: A. one or more pseudotyping elementson its surface, wherein the one or more pseudotyping elements arecapable of binding to a T cell and/or an NK cell and facilitatingmembrane fusion of the recombinant retrovirus thereto; B. apolynucleotide comprising one or more transcriptional units operativelylinked to one or more promoters active in T cells and/or NK cells,wherein the one or more transcriptional units encode i.a firstengineered signaling polypeptide comprising a lymphoproliferativeelement, wherein the lymphoproliferative element promotes proliferationand/or survival of T and/or NK cells; and ii.a second engineeredsignaling polypeptide comprising a chimeric antigen receptor comprisingan antigen-specific targeting region, a transmembrane domain, and anintracellular activating domain; and C. an activation element on itssurface, wherein the activation element is fused to a membraneattachment sequence, and wherein the activation element is capable ofbinding to a T cell and/or NK cell and is not encoded by apolynucleotide in the recombinant retrovirus.
 29. The composition ofclaim 28, wherein the composition is a pharmaceutical composition. 30.The composition of claim 28, wherein the composition is in a container.31. The composition of claim 30, where in the container is part of akit.
 32. The composition of claim 28, wherein the purified replicationincompetent recombinant retroviruses are the product of a purificationprocess comprising concentration or diafiltration, or both concentrationand diafiltration.
 33. The composition of claim 28, wherein thepurification process comprises tangential flow filtration.
 34. Thecomposition of claim 28, wherein the purified replication incompetentrecombinant retroviruses are the product of a purification processcomprising centrifugation, polyethylene glycol (PEG) precipitation,and/or depth filtration.
 35. The composition of claim 28, wherein thepurification process comprises depth filtration and tangential flowfiltration.
 36. A composition comprising isolated replicationincompetent recombinant retroviruses, wherein the isolated replicationincompetent recombinant retroviruses comprise: A. one or morepseudotyping elements on its surface, wherein the one or morepseudotyping elements are capable of binding to a T cell and/or an NKcell and facilitating membrane fusion of the recombinant retrovirusthereto; B. a polynucleotide comprising one or more transcriptionalunits operatively linked to one or more promoters active in T cellsand/or NK cells, wherein the one or more transcriptional units encodeiii.a first engineered signaling polypeptide comprising alymphoproliferative element, wherein the lymphoproliferative elementpromotes proliferation and/or survival of T and/or NK cells; and iv.asecond engineered signaling polypeptide comprising a chimeric antigenreceptor comprising an antigen-specific targeting region, atransmembrane domain, and an intracellular activating domain; and C. anactivation element on its surface, wherein the activation element isfused to a membrane attachment sequence, and wherein the activationelement is capable of binding to a T cell and/or NK cell and is notencoded by a polynucleotide in the recombinant retrovirus.
 37. Thecomposition of claim 28, wherein the purified replication incompetentrecombinant retroviruses are free of serum components.
 38. Thecomposition of claim 28, wherein the purified replication incompetentrecombinant retroviruses are free of non-human animal media components.39. The composition of claim 36, wherein the isolated replicationincompetent recombinant retroviruses are free of serum components. 40.The composition of claim 36, wherein the isolated replicationincompetent recombinant retroviruses are in a buffer.
 41. Thecomposition of claim 40, wherein the buffer is phosphate bufferedsaline.
 42. The composition of claim 41, wherein the composition furthercomprises lactose.
 43. The composition of claim 36, wherein the isolatedreplication incompetent recombinant retroviruses are in a serum-freemedium.
 44. The composition of claim 28, wherein the purifiedreplication incompetent recombinant retroviruses are in a buffer. 45.The composition of claim 44, wherein the buffer is phosphate bufferedsaline.
 46. The composition of claim 45, wherein the composition furthercomprises lactose.
 47. The composition of claim 28, wherein the purifiedreplication incompetent recombinant retroviruses are in a scrum-freemedium.
 48. The composition of claim 28, wherein the purifiedreplication incompetent recombinant retroviruses are in a frozen state.